Genex Altfragen 2.0

Genexpression Altfragenausarbeitung
Decker & Moll – WiSe 2018 / 2019

Altfragensammlung bis 2018, beantwortet mit den Folien sowie den Vorlesungsmitschriften. Die Fragen sind aus dem scheinbar nicht
enden wollenden Altfragenkonglomerat so ausgewählt, dass möglichst der ganze Stoff abgedeckt ist (v.a. beim Decker). Da es von
der Moll derzeit noch nicht wirklich Altfragen gibt, habe ich einige scheinbar beliebte Altfragen vom Bläsi noch dazu genommen.
Die Fragen sind nach bestem Wissen und Gewissen ausgearbeitet, trotzdem übernehme ich keine Gewähr bezüglich der Korrektheit.
Viel Erfolg beim Lernen!
Katharina J. Kases
Decker: Multiple Choice
SIGMA-FAKTOREN SIND TEIL DER RNA POLYMERASE IN DER ELONGATIONSPHASE.

a. richtig
b. falsch
By associating to the sigma factor the RNA polymerase forms a holoenzyme which binds un-
specifically to the DNA. The bound sigma factor allows the RNA polymerase to perform a one-di-
mensional search for the promoter sequence. After the promoter is bound, first a closed complex
forms, followed by the conformational transition to an open complex. During the promoter clear-
ance the sigma factor dissociates so that only the core enzyme proceeds to elongation.
DER ELEKTROPHORETIC MOBILITY SHIFT ASSAY ZEIGT DIE BINDUNG VON TRANSKRIPTIONS-
FAKTOREN AUS KERNEXTRAKTEN AN REGULATORISCHEN DNA SEQUENZEN.
a. richtig
b. falsch
The electrophoretic mobility shift assay (EMSA) can be applied to determine the binding of tran-
scription factors to the DNA. For this purpose, radioactively labelled DNA fragments carrying a TF
binding site are incubated with a nuclear extract of transcription factors. Binding of the transcrip-
tion factors to the DNA fragment is monitored by electrophoresis on a polyacrylamide gel. By as-
sociation of the proteins to the DNA a complex is formed, leading to a shift in the polyacrylamide
gel.
OPERONS FÜHREN ZUR ERZEUGUNG POLYCISTRONISCHER RNA.

a. richtig
b. falsch
Transcriptional control in bacteria is mediated by the operon, an array of genes characteristic of

bacteria. These genes are positioned in direct succession on the DNA, under coordinate regula-
tion and are therefore transcribed into one polycistronic mRNA.  
1 of 56
ÖFFNEN EINER PROMOTERSTRUKTUR FÜHRT ZUM ERLIEGEN DER TRANSKRIPTION.
a. richtig
b. falsch
RNA synthesis commences by RNA polymerase binding to the promotor containing the transcrip-
tion start and formation of the closed complex. The open complex formation describes the open-
ing of the DNA duplex within the RNA polymerase, leading to the so-called transcription bubble
formed by the separated DNA strands. This allows transcription of the template (non-coding)
strand during (promotor clearance /) elongation.
2-KOMPONENTENSYSTEME ÜBERTRAGEN PHOSPHATGRUPPEN.

a. richtig
b. falsch
The phoR-phoB two component system regulates genes that maintain intracellular phosphate
levels. The sensor protein phoR is a membrane-associated kinase, recognising certain metabo-
lites and transferring phosphates onto the response regulator phoB; subsequently, target genes
can be transcribed. Another example of a two component system are NtrB and NtrC, controlling
the transcription of glutamine synthase, whose gene responds to a decrease of organic nitrogen.
Although the RNA polymerase is permanently associated to the glnA (synthase) gene, it cannot
open the promoter. Hence, the activity of the RNA polymerase is dependent on phosphorylation
of NtrC by NtrB, which (when phosphorylated) can bind to the DNA, interact with the RNA poly-
merase and subsequently the promoter can be opened.
EUKARYOTISCHE INITIATIONSKOMPLEXE FÜR ALLE RNA POLYMERASEN ENTHALTEN TBP.

a. richtig
b. falsch
Recent studies of the three eukaryotic transcription machineries revealed that all initiation com-
plexes share a conserved core. This core consists of the RNA polymerase (I, II, or III), the TATA
box-binding protein (TBP), and transcription factors TFIIB, TFIIE, and TFIIF (for Pol II) or proteins
structurally and functionally related to parts of these factors (for Pol I and Pol III). The conserved
core initiation complex stabilises the open DNA promoter complex and directs initial RNA synthe-
sis. The periphery of the core initiation complex is decorated by additional polymerase-specific
factors that account for functional differences in promoter recognition and opening, and gene
class-specific regulation.
https://www.cell.com/action/showPdf?pii=S1097-2765%2812%2900089-5
DER GENERELLE TF TFIIB INTERAGIERT SOWOHL MIT DER RNA POLYMERASE ALS AUCH MIT
DEM TF TFIID.
a. richtig
b. falsch
The formation of the initiation complex of RNA polymerase II dependent promotors is usually in-
duced by the binding of TFIID (containing TBP and TBP associated factors, TAFs) to the TATA
box. The initiation complex comprises RNA Pol II, TFIID, TFIIB, TFIIF and TFIIA. Transcription fac-
tor IIH phosphorylates RNA Pol II at its carboxy-terminal domain (CTD) to enable elongation. To
2 of 56
accelerate reinitiation of transcription a scaffold complex remains on the DNA. Thereby energy is
saved as reinitiation is less energy consuming than the first initiation.
DER NUCLEAR RUN-ON ASSAY MISST DIE INITIATION DURCH RNA POLYMERASE.
a. richtig
b. falsch
The nuclear run-on assay is an out-dated method to analyse transcriptional control by RNA poly-
merase II. This method tries to determine wether mRNA concentrations increase due to tran-
scriptional control or increased mRNA stability. A nuclear run-on assay is conducted to identify
cell-specific genes that are being transcribed at a certain time point. In order to achieve this, nu-
clei are isolated and incubated with radioactively labeled nucleotides; genes in the process of be-
ing transcribed incorporate the radioactive nucleotides during transcription. To this reaction ex-
cess target gene DNA is added and the relative transcription of the target gene is detected by hy-
bridisation of extracted RNA to gene specific probes on a blot.
https://en.wikipedia.org/wiki/Nuclear_run-on
ALLE EUKARYOTEN RNA POLYMERASEN WERDEN IN DER C-TERMINALEN DOMÄNE PHOSPHO-

RYLIERT.
a. richtig
b. falsch
Initiation of RNA Pol II-dependent promoters transcription is achieved by the binding of the gen-
eral transcription factor TFIID (containing TBP and TBP-associated factors, TAFs) to the TATA box
(core promoter). TFIIH phosphorylates RNA Pol II at serine 5 (Ser5) on its carboxy-terminal domain
(CTD), containing up to 52 heptapeptide-repeats of amino acids which can be phosphorylated.
This allows promoter clearance, the RNA Pol II transcribes a small fragment of the gene, back-
tracks and repeats transcription. However, no further transcription is possible because of the
elongation block. Hence, a second phosphorylation at serine 2 (Ser2) in the heptapeptide-repeats
of CTD by the elongation factor P-TEFb is needed in order to allow transcriptional elongation.
Therefore, the RNA Polymerase II transcription cycle is dependent on phosphorylation and de-
phosphorylation of the CTD, as its phosphorylation by TFIIH and P-TEFb causes association of
proteins that participate in mRNA processing.
In contrast, initiation of transcription by RNA polymerase I is mediated by the binding of upstream

binding factors (UBF) to the UCE sequence, leading to the formation of the initiation complex at
the core promoter. 5S rRNA transcription by RNA polymerase III is initiated from within the gene
body, containing two binding motifs for the general transcription factor TFIIIC, which subsequently
will interact with TFIIIB through TBP.
ELONGATION DER TRANSKRIPTION ERFORDERT VOLLSTÄNDIGE ENTFERNUNG DER NUKLEO-

SOMEN.
a. richtig
b. falsch
Nucleosomes overlapping the transcription start or transcription factor binding sites need to be
moved in order for transcription factors to be able to bind to the DNA. Chromatin remodelling
complexes can assist in chromatin assembly by moving already deposited histone octamers,
3 of 56
generating room for additional disposition. Remodeller complexes (e.g. SWI/SNF or ISWI) have
two distinct functions, either they lead to site exposure or they enable an altered composition of
nucleosomes. Binding sites can be exposed via three different pathways: chromatin remodellers
enable repositioning of the binding site by nucleosomal sliding, nucleosomal ejection (eviction of
the nucleosomal complex) or localised unwrapping of the DNA. The elongation factor FACT (facili-
tator of transcription) displaces the H2A/H2B dimer from the nucleosome, thereby loosens the
contact between DNA and histone, allowing transcription. Altered composition is achieved either
by dimer exchange whereby a H2A/H2B dimer is exchanged with a histone variant (interplay of
chaperones and chromatin remodellers) or dimer ejection (removal of the H2A/H2B dimers).
SOWOHL PROKARYOTEN ALS AUCH EUKARYOTEN VERFÜGEN ÜBER TRANSKRIPTIONSFAK-

TOREN DIE AUF SCHWERMETALLE REAGIEREN.
a. richtig
b. falsch
The MerR family is a group of transcriptional activators with similar N-terminal helix-turn-helix
DNA binding regions and C-terminal effector binding regions that are specific to the effector
recognised. MerR-like regulators have been found in a wide range of bacterial genera, but not yet
in archaea or eukaryotes. However, there are various metal-responsive transcription factors that
regulate iron, zinc, and copper homeostasis also in eukaryotic cells as iron, copper, and zinc are
all essential nutrients. Organisms need to maintain cytoplasmic metal concentrations at a nontox-
ic level that is sufficient for growth, which is achieved by heavy-metal dependent transcription
factors. These factors are able to sense changes in metal concentrations and coordinate the ex-
pression of genes that are involved in the acquisition, distribution, and use of metals.
https://www.ncbi.nlm.nih.gov/pubmed/12829265
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC329510/
TRANSKRIPTIONSFAKTOREN IN DER LEBER REAGIEREN AUF DIE GEGENWART VON LAKTOSE.

a. richtig
b. falsch
Lactase is encoded by a single genetic locus (LCT) on chromosome 2. It is expressed exclusively

by mammalian small intestine enterocytes and in very low levels in the colon during fetal devel-
opment. Mature human lactase consists of a single 160-kDa polypeptide chain that localises to
the brush border membrane of intestinal epithelial cells. It is oriented with the N-terminus outside
the cell and the C-terminus in the cytosol. Although Humans are born with high levels of lactase
expression, in most of the world’s population, lactase transcription is down-regulated after wean-
ing. This results in diminished lactase expression in the small intestine, which causes the common
symptoms of adult-type hypolactasia, or lactose intolerance. https://en.wikipedia.org/wiki/Lactase
Dietary fatty acids are well-established regulators of hepatic transcription factors, however emerg-
ing evidence indicates that endogenously generated fatty acids are equally important in control-
ling transcription factors in the context of glucose and lipid homeostasis.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940310/pdf/nihms557375.pdf
4 of 56
DIMERISIERUNG VON STAT WIRD DURCH TYROSINPHOSPHORYLIERUNG REGULIERT.
a. richtig
b. falsch
Signal transducer and activator of transcription (Stat) transcription factors are activated by JAK
which phosphorylates a specific tyrosine residue within STAT, hence facilitating dimerisation via
their SH2 domains.
TF BINDEN AN DIE RNA POLYMERASE.

a. richtig
b. falsch
A transcription factor (TF) is a protein that controls the rate of transcription of genetic information
from DNA to messenger RNA, by binding to a specific DNA sequence. TFs work alone or with
other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the re-
cruitment of RNA polymerase to specific genes. A defining feature of transcription factors is that
they contain at least one DNA-binding domain (DBD), which attaches to a specific sequence of
DNA adjacent to the genes that they regulate. TFs are grouped into classes based on their DBDs.
Other proteins such as co-activators, chromatin remodellers, histone acetyltransferases (HATs),
histone deacetylases (HDACs), kinases, and methylases are also essential to gene regulation, but
lack DNA-binding domains, and therefore are not transcription factors. https://en.wikipedia.org/wiki/
Transcription_factor
Only some transcription factors such as TFIIB can bind to RNA polymerase II directly, others rely
on the mediator complex (bound to CTD of RNA Pol II) which mediates between RNA polymerase
II and transcription factors, thereby coordinating transcriptional initiation and elongation.
MONOMERE TRANSKRIPTIONSFAKTOREN BINDEN PALINDROMISCHE DNA.

a. richtig
b. falsch
Some nuclear hormone receptors are constantly bound to DNA, acting as transcriptional repres-
sors as long as no ligand is bound. Upon binding of their ligand, nuclear hormone receptors un-
dergo a conformational change whereby they are now acting as a transcriptional activator. Nu-
clear hormone receptors associate with DNA either as homodimers, binding inverted (palin-
dromic) repeats, or as heterodimers binding direct repeats.
ALLE BAKTERIELLEN TF ÜBEN NEGATIVE TRANSKRIPTIONSKONTROLLE AUS.

a. richtig
b. falsch
A transcription factor is a protein that controls the rate of transcription of genetic information from
DNA to messenger RNA, by binding to a specific DNA sequence. Activators are transcription fac-
tors promoting the recruitment of RNA polymerase, whereas repressors are transcription factors
blocking RNA Pol recruitment. A defining feature of TFs is that they contain at least one DNA-
binding domain (DBD), which attaches to a specific sequence of DNA adjacent to the genes that
they regulate. https://en.wikipedia.org/wiki/Transcription_factor
5 of 56
Bacterial activators and repressors have at least three distinct mechanisms through which they
can influence gene expression. For instance, Class I activators can directly associate with the α-
subunit of RNA polymerase and thereby facilitate RNA Pol binding to DNA. In contrast, Class II
activators partly overlap the promotor sequence and interact with the RNA Pol’s ß-subunit. Fur-
thermore, some activators induce a conformational change enabling interaction between RNA
polymerase and DNA. Repressors can suppress transcription by steric hindrance, DNA looping or
modulation of an activator. The latter is an example for the lac repressor which does not inhibit
RNA polymerase binding but further steps of transcriptional activation such as opening of the
operon.
LACTOSE IST EIN KOAKTIVATOR FÜR DEN LAC-REPRESSOR.

a. richtig
b. falsch
Regulation of the lac mRNA depends on the addition of the inducer allolactose (a derivative of the
substrate lactose). Presence of lactose results in a rapid induction of lac mRNA and subsequently
(after a short lag) the synthesis of the enzymes, whereas removal of the inducer is followed by the
rapid cessation of synthesis. The lac repressor (lacI) is active as a dimer, possessing a helix-turn-
helix motif with a palindromic DNA binding site, allowing binding of DNA in both directions. One
helix of the repressor is responsible for base-specific contact with DNA, the other binds to the
DNA backbone. Allolactose induces a conformational change in the lac repressor, whereby the
repressor is no longer able to bind to DNA.
ALLE TF DER NUCLEAR (HORMONE) RECEPTOR FAMILIE BINDEN AN DIRECT REPEAT DNA.
a. richtig
b. falsch
Binding sites for nuclear hormone receptors form either inverted repeats (palindromes) or direct
repeats. Homodimers of glucocorticoids and sex hormones bind to palindromic sequences,
whereas heterodimeric receptors of the RXR family bind to direct repeats. Those direct repeats
are sequentially always the same but differ in the spacing between them. Thereby nuclear hor-
mone receptors can recognise and bind to each sequence specifically.
DIE ZINK FINGER STRUKTUR KONTAKTIERT DIE KLEINE FURCHE (MINOR GROOVE) DER DNA.
a. richtig
b. falsch
Zinc finger structures are present in a large number of mammalian proteins, for instance enabling
DNA binding or protein interactions. Zinc fingers can either form a complex with two cysteines
and two histidines (C2H2) or with four cysteines (C4 finger). C2H2 proteins contain three or more
fingers and bind DNA as monomers whereas C4 proteins contain two fingers and bind as dimers.
The zinc finger structure describes a compact array of ß-sheets and α-helices through the coordi-
nation of a Zn2+ atom. The Zinc finger α-helices interact with DNA in its major groove.
6 of 56
LEUCINE ZIPPER SIND IN EUKARYOTEN TF MIT EINER BASISCHEN REGION ASSOZIIERT.
a. richtig
b. falsch
Leucine zippers are examples of a coiled-coil structure formed by amphipathic helices; they serve
to homo- or heterodimerise proteins. Transcriptional regulators combine leucine zippers with a
basic region, containing many positively charged amino acids, which enables binding of DNA.
DER GENERELLE TRANSKRIPTIONSFAKTOR TBP ÄNDERT DIE DNA STRUKTUR.

a. richtig
b. falsch
Some eukaryotic promoters of mRNA genes contain a TATA box motif which is recognised and
bound by the TATA box binding protein (TBP). Upon binding the TATA box, the TBP monomer in-
duces a bend in the DNA around the initiation site.
EUKARYOTISCHE TF KÖNNEN DIE ELONGATION DER TRANSKRIPTION REGULIEREN.

a. richtig
b. falsch
Initiation of RNA Pol II-dependent promoters transcription is achieved by the binding of the gen-
eral transcription factor TFIID (containing TBP and TBP-associated factors, TAFs) to the TATA box
(core promoter). TFIIH phosphorylates RNA Pol II at serine 5 (Ser5) of its carboxy-terminal domain
(CTD), containing up to 52 heptapeptide-repeats of amino acids which can be phosphorylated.
This allows promoter clearance, the RNA Pol II transcribes a small fragment of the gene, back-
tracks and repeats transcription. However, no further transcription is possible because of the
elongation block. Hence, a second phosphorylation at Ser2 in the heptapeptide-repeats of CTD
by the elongation factor P-TEFb is needed in order to allow transcriptional elongation. Therefore,
the RNA Polymerase II transcription cycle is dependent on phosphorylation and dephosphoryla-
tion of the CTD, as its phosphorylation by TFIIH and P-TEFb causes association of proteins that
participate in mRNA processing.
ALLE EUKARYOTISCHEN PROMOTOREN HABEN EINE TATA-BOX.

a. richtig
b. falsch
The TATA box is considered a non-coding DNA sequence (cis-regulatory element), containing a
consensus sequence characterised by repeating T and A base pairs. The TATA box is a compo-
nent of some eukaryotic promoters and functions as the transcription initiation site in TATA box
containing genes. The TATA box can be bound not only by TBP but also some transcription fac-
tors. Gene transcription by RNA polymerase II depends on the regulation of the core promoter by
long-range regulatory elements such as enhancers and silencers.
https://en.wikipedia.org/wiki/TATA_box 
7 of 56
DER GENERELLE TRANSKRIPTIONSFAKTOR TFIID ENTHÄLT EINE HISTON-METHYLTRANSFERASE.
a. richtig
b. falsch
The general transcription factor TFIID is a large protein complex offering contact surfaces for oth-
er transcription factors. TFIID contains not only TBP but also 13 to 14 TBP-associated factors
(TAFs) which determine the specificity of TFIID for particular DNA sequences, its association with
TBP, contact with other transcription factors as well as histone acetylation. Binding of the gener-
al transcription factor TFIID to the TATA box motif (core promoter) usually initiates transcription of
RNA Pol II-dependent promoters.
DIE REIHENFOLGE IN DER DIE DER GENERELLEN TRANSKRIPTIONSFAKTOREN AN DEN PRO-

MOTER BINDEN WURDE MITTELS EMSA ERMITTELT.
a. richtig
b. falsch
Sequential formation of an initiation complex from general transcription factors at the site of the
promoter can be investigated with an electrophoretic mobility shift assay (EMSA). For this pur-
pose, a radioactively labelled DNA fragment carrying a promoter is incubated with a combination
of various transcription factor. Binding of the proteins to the DNA fragment is monitored by elec-
trophoresis on a polyacrylamide gel. With addition of each component a larger complex is formed,
provided that the component are added in the correct order to assemble an active transcription
complex.
DER GENERELLE TRANSKRIPTIONSFAKTOR TBP IST AN DER INITIATION ALLER EUKARYOTISCH-

ER RNA-POLYMERASEN BETEILIGT.
a. richtig
b. falsch
Recent studies of the three eukaryotic transcription machineries revealed that all initiation com-
plexes share a conserved core. This core consists of the RNA polymerase (I, II, or III), the TATA
box-binding protein (TBP), and transcription factors TFIIB, TFIIE, and TFIIF (for Pol II) or proteins
structurally and functionally related to parts of these factors (for Pol I and Pol III). The conserved
core initiation complex stabilises the open DNA promoter complex and directs initial RNA synthe-
sis. The periphery of the core initiation complex is decorated by additional polymerase-specific
factors that account for functional differences in promoter recognition and opening, and gene
class-specific regulation.
https://www.cell.com/action/showPdf?pii=S1097-2765%2812%2900089-5
CHROMATIN REMODELLING KOMPLEXE ENTHALTEN ATPASEN.

a. richtig
b. falsch
All chromatin remodelling complexes such as SWI/SNF or ISWI have to contain proteins, i.e. AT-
Pases, which are able to bind histones and convert energy (ATP to ADP) to enable chromatin re-
modelling. ATPases alone can facilitate sliding and transfer mechanisms, however the transfer of
nucleosomes onto free DNA needs chromatin remodeller complexes. 
8 of 56
DER MEDIATOR KOMPLEX VERMITTELT DIE BINDUNG ZWISCHEN CHROMATIN REMODELLING
KOMPLEXEN UND DER RNA POLYMERASE.
a. richtig
b. falsch ?
Association of chromatin remodelling complexes and histone modifying complexes with chro-
matin occurs either through reading of established modifications or through association with tran-
scription factors (or both). Mediator is a multiprotein complex that functions as a transcriptional
coactivator in all eukaryotes, mediating interactions between RNA polymerase II and transcription
factors. The mediator complex comprises subunits which directly bind RNA Pol II and subunits
which bind transcription factors; its core interacts with the CTD of RNA polymerase II.
Transcriptional activation of the Gal1 gene by Gal4 is induced as soon as galactose is present in
the cell. In absence of galactose, the Gal1 gene is repressed by its bound transcriptional activator
Gal4 which is associated with a repressor protein. In presence of galactose, the transcriptional
activator recruits a HAT, a chromatin remodelling complex as well as a mediator complex. After
histone acetylation and nucleosome remodelling, a pre-initiation complex containing the remod-
eller, HAT, the mediator and RNA Pol II can be formed.
Decker Part 2, p. 35
Decker Part 2, p. 76
TRANSKRIPTIONSFAKTOREN KÖNNEN HATS UND HDACS MIT DEM CHROMATIN VON PROMO-
TOREN ASSOZIIEREN.
a. richtig
b. falsch
Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are recruited to their target
promoters through physical interactions with sequence-specific transcription factors. They usually
function within a multisubunit complex in which the other subunits are necessary for them to
modify histone residues around the binding site. Wikipedia: histone acetyltransferase
Histones H2 and H4 possess long N-terminal unstructured regions (“histone tails”) which are pro-
truding from the nucleosome and therefore can be post-translationally modified. Acetylation by
HATs neutralise the positive charge of lysine residues thereby loosening the interaction between
DNA and nucleosome, increasing the accessibility of DNA for transcription factors. HDACs exert
the opposite effect by removing acetyl groups from lysines. Acetylation indicates transcriptional
activity, whereas methylated lysines indicate that DNA is inactive. Other amino acids which can be
post-translationally modified include arginine (methylation) and serine or threonine which can be
phosphorylated.
CHIP BZW. CHIP-SEQ. WERDEN ZUM ERSTELLEN VON DURCH HISTON-MODIFIKATIONEN

CHARAKTERISIERTEN CHROMATIN LANDSCHAFTEN EINGESETZT.
a. richtig
b. falsch
Histone variants, histone modifiers / modifications and transcription factors define the chromatin
landscape. ChIP-Seq technology combines chromatin immunoprecipitation (IP) with massive par-
allel sequencing and bioinformatic alignment of short sequence reads to genomes. Thereby
analysis of histone modifications is possible since antibodies used for precipitation only bind to
histones if certain modifications are present. The so-called histone code can be determined by
9 of 56
associating specific modifications with their position and a particular, correlating modified func-
tion.
CHROMATIN REMODELLING KOMPLEXE KÖNNEN HISTONE IN EINEM NUKLEOSOM AUS-

TAUSCHEN.
a. richtig
b. falsch
moved in order for transcription factors to be able to bind to DNA. Chromatin remodelling com-
plexes can assist in chromatin assembly by moving already deposited histone octamers, generat-
ing room for additional disposition. Remodeller complexes (e.g. SWI/SNF, ISWI) have two distinct
functions, either they lead to site exposure or they enable an altered composition of nucleosomes.
Binding sites can be exposed via three different pathways: chromatin remodellers enable reposi-
tioning of the binding site by nucleosomal sliding, nucleosomal ejection (eviction of the nucleoso-
mal complex) or localised unwrapping of the DNA. An altered composition is achieved either by
dimer exchange whereby a H2A/H2B dimer is exchanged with a histone variant (interplay of
NUKLEOSOMEN BESTEHEN AUS EINEM H3/H4 TETRAMER UND AUS ZWEI H2A/H2B DIMEREN.
a. richtig
b. falsch
AKTIVE GENE SIND AM AUSTAUSCH VON HISTONEN GEGEN HISTONVARIANTEN ERKENNBAR.

a. richtig
b. falsch
The ATP-dependent chromatin remodeller complexes such as SWI/SNF or ISWI have two distinct
functions: exposing binding sites on DNA for transcription factors and altering the composition of
nucleosomes. If nucleosomes are overlapping the transcription start they need to be moved either
by repositioning, ejection or unwrapping of the DNA, to allow binding of transcription factors to
DNA. Altering the composition of nucleosomes is a second function, whereby remodellers,
through cooperation with chaperones, exchange or remove H2A/H2B dimers.
Histone variants are involved in epigenetic properties of active genes; both H3.3 and H2A.Z are
enriched at transcriptionally active loci. The dynamic behaviour of chromatin leads to the realisa-
tion that transcription, chromatin remodelling, and histone modification might be coupled to nu-
cleosome assembly and disassembly. In addition to these universal processes, histone variants
are also involved in particular epigenetic phenomena. In the case of the mammalian X chromo-
some, three different H2A variants have been recruited to participate in silencing or activation of
genes for purposes of germline inactivation or dosage compensation.
https://cshperspectives.cshlp.org/content/7/1/a019364.full.pdf
10 of 56
BEI DER ELONGATION WERDEN NUCLEOSOMEN VOLLSTÄNDIG AUS DEM TRANSKRIBIERTEN
BEREICH ENTFERNT.
a. richtig
b. falsch
The elongation factor FACT (facilitator of transcription) displaces the H2A/H2B dimer from the nu-
cleosome, thereby looses the contact between DNA and histone, allowing transcription. In addi-
tion, FACT acts as a histone-chaperone to reinstall the H2A/H2B dimer.
AKTIVE PROMOTOREN SIND AN IHREM HISTON-METHYLIERUNGSMUSTER ERKENNBAR.

a. richtig
b. falsch
Acetylation of histones at lysine residues leads to transcriptional activation, whereas phosphory-

lation of serine or threonine mostly leads to mitosis, DNA repair or even apoptosis. However,
methylation of lysine or arginine can both be linked to transcriptional activation as well as silenc-
ing.
TRANSKRIBTIONSSTART IST GENERELL FREI VON HISTONEN.

a. richtig
b. falsch
moved in order for transcription factors to be able to bind to the DNA. Chromatin remodelling
complexes can assist in chromatin assembly by moving already deposited histone octamers,
generating room for additional disposition. Remodeller complexes (e.g. SWI/SNF, ISWI) have two
distinct functions, either they lead to site exposure or they enable an altered composition of nu-
cleosomes. Binding sites can be exposed via three different pathways: chromatin remodellers en-
able repositioning of the binding site by nucleosomal sliding, nucleosomal ejection (eviction of the
nucleosomal complex) or localised unwrapping of the DNA. Altered composition is achieved either
by dimer exchange whereby a H2A/H2B dimer is exchanged with a histone variant (interplay of
MIT HILFE VON REPORTER GENE ASSAYS KANN MAN DIE BINDUNG VON TRANSKRIPTIONSFAK-
TOREN AN EINEN PROMOTER BESTIMMEN.
a. richtig
b. falsch
Reporter gene assays allow the determination of promoter activity by introducing reporter genes
which are not naturally present in the cells used for the assay. Those genes are mostly of bacterial
or invertebrate origin; frequently used reporter genes include CAT (chloramphenicol-acetyl-trans-
ferase), ß-galactosidase, luciferase and GFP. Promoter bashing / deletion mapping identify func-
tional elements of promoter-DNA essential for promoter activity as well as transcription factor
binding sites.
Reporter gene assays are invaluable for studying regulation of gene expression, both by cis-acting
factors (gene regulatory elements) and trans-acting factors (transcription factors or exogenous
11 of 56
regulators). Furthermore, reporter gene systems enable the use of pathway-specific, tissue-spe-
cific, or developmentally regulated gene promoters as biomarkers for specific events processes.
https://www.thermofisher.com/ng/en/home/references/gibco-cell-culture-basics/transfection-basics/reporter-gene-as-
says.html
ELONGATION DER TRANSKRIPTION BEGINNT NACHDEM HISTONE PHOSPHORYLIERT WURDEN.

a. richtig
b. falsch
Acetylation of histones at lysine residues leads to transcriptional activation, whereas phosphory-

lation of serine or threonine mostly leads to mitosis, DNA repair or even apoptosis. Another post-
translational modification, methylation of lysine or arginine, can both be linked to transcriptional
activation as well as silencing.
CHROMATINIMMUNPRÄZIPITATION / SEQUENZIERUNG BESTIMMT DIE POSITION VON NUKLEO-

SOMEN INNERHALB EINES GENS.
a. richtig
b. falsch
ChIP-Seq technology combines chromatin immunoprecipitation (IP) with massive parallel se-
quencing and bioinformatic alignment of short sequence reads to genomes. Thereby analysis of
histone modifications is possible since antibodies used for precipitation only bind to histones if
certain modifications are present. Hence, the so-called histone code can be determined by asso-
ciating specific modifications with their position and a particular, correlating modified function.
CHROMATIN HUBS KÖNNEN TRANSKRIBIERTE UND NICHTTRANSKRIBIERTE BEREICHE DES

GENOMS TRENNEN.
a. richtig ?
b. falsch
Insulator (Ins) and locus control regions (LCR) separate distinct chromosomal regions. Insulator
sequences insulate euchromatin (actively transcribed genomic regions) from heterochromatin
(non-transcribed genomic regions) as the latter would otherwise spread into the euchromatin. In a
broader definition, insulator elements form boundaries between genomic regions with different
structural and functional properties. Locus control regions on the other hand, determine by inter-
action with the promoter or enhancer which genes within a chromosomal domain can be tran-
scribed. Chromatin hubs are produced by the interaction between locus control regions with in-
sulator sequences and often involve CTCF.
PROTEINE DER TRITHORAX-GRUPPE INDUZIEREN HETEROCHROMATIN.

a. richtig
b. falsch
Epigenetic control of cellular (transcriptional) memory in Drosophila melanogaster: polycomb

complexes (PcG) are readers and writers of histone modifications and therefore involved in the
formation and propagation of heterochromatin. Chromatin states such as euchromatin or hete-
12 of 56
rochromatin can be passed on to descendant cells if the chromatin state is mitotically stable. The
interaction between polycomb complexes and the DNA is controlled by transcription factors:
transcriptional activators prevent binding of polycomb complexes to the DNA, whereas transcrip-
tional repressors stimulate polycomb binding. Polycomb complexes heterochromatise euchro-
matin e.g. by trimethylation of H3 (H3K9Me / H3K27Me). PRC1 for instance, recognises K27Me
and transfers ubiquitins onto the residue, thereby interfering with transcription. PRC2 recognises
the methylated K27 and confers the same modification onto a neighbouring lysine, hereby facili-
tating the spreading the heterochromatin. Trithorax protein complexes (Trx) are recruited by tran-
scriptional activators to antagonise polycomb complexes. Both trxG and PcG families include
proteins that covalently modify histones and proteins that non-covalently modify chromatin. Cova-
lent modifications on histones can increase binding by non-covalently modifying complexes
which has the potential to lead to further covalent modifications. Thereby iterative cycles of estab-
lishment and recognition of covalent modifications can be induced.
HISTON DEACETYLIERUNG BEWIRKT DIE METHYLIERUNG DER DNA.

a. richtig
b. falsch
Histone acetylation by HATs and deacetylation by HDACs at lysine residues within the N-terminal
tail protruding from the histone core of the nucleosome are processes essential to gene regula-
tion. Acetylated histones, octameric proteins that organise chromatin into nucleosomes and ulti-
mately higher order structures, represent a type of epigenetic marker within chromatin. Acetylation
removes the positive charge of the histones, thereby decreasing the interaction of the N-termini of
histones with the negatively charged phosphate groups of DNA. As a consequence, the con-
densed chromatin is transformed into a more relaxed structure that is associated with greater lev-
els of gene transcription (euchromatin). Condensation can be brought about by processes includ-
ing deacetylation (reversing to a tightly packed chromatin structure) and methylation.
https://en.wikipedia.org/wiki/Histone_acetylation_and_deacetylation
In case of CpG methylation, the protein MeCP silences chromatin by recruiting HDAC and methyl-
transferase complexes. Active transcription of acetylated histones transitions into silencing by
methylation through Dnmts, followed by further methylation and deacetylation through recruitment
of HDAC and HKMT by MeCP. This process describes a hypothetical transition between an active,
non-methylated gene promoter and a repressed promoter whose silence is due to DNA meth-
ylation mediated by MeCP2.
Decker, Part 2, p. 109
UNTERSCHIEDLICHE SIGMA FAKTOREN DIRIGIEREN RNA POLYMERASE ZU SPEZIFISCHEN GEN-

GRUPPEN.
c. richtig
d. falsch
Sigma factors allow prokaryotic RNA polymerase to discriminate between specific promotors and
random DNA sequences. Diverse sigma factors direct coordinate expression of defined groups of
genes. E.g. SPO1 phage directs the switch from early to late gene expression by association of its
RNA polymerase to various sigma factors which are specific either for early, middle or late gene
expression. 
13 of 56
NUKLEOSOMEN ENTHALTEN ANHÄUFUNGEN NEGATIV GELADENER AMINOSÄUREN ZUR INTER-
AKTION MIT DNA.
a. richtig
b. falsch
A nucleosome comprises an octamer of histones consisting of one H3/H4 tetramer and two H2A/
H2B dimers. Neighbouring nucleosomes are spaced with linker DNA which is bound by histone
H1 causing compaction of nucleosomal DNA. The association of DNA with the nucleosome is fa-
cilitated by electrostatic interactions between the (positively charged) histone octamer and the
(negatively charged) DNA.
DNA SCHLAUFEN KÖNNEN ZUR GENEXPRESSION IN BAKTERIEN BEITRAGEN.

a. richtig
b. falsch
The glutamine synthase gene responds to a decrease of organic nitrogen; NtrcB and NtrcC form a
two component system to regulate nitrogen fixating genes. RNA polymerase complexed with
sigma factor 54 is permanently associated with the promoter of the glnA (glutamine synthase)
gene, but cannot open the promoter. Phosphorylation of NtrC by NtrB causes it to bind to DNA,
inducing an ATP-dependent conformational change in DNA enabling direct contact of NtrC and
the RNA polymerase. Interaction of NtrC and the RNA polymerase induces formation of a DNA
loop. Thereby the glnA promoter is activated, an open complex is formed and transcription is
possible.
LOCUS CONTROL REGIONS KÖNNEN AN DER BILDUNG VON CHROMATIN HUBS BETEILIGT SEIN.
a. richtig
b. falsch
scribed. Chromatin hubs are produced by the interaction between locus control regions with insu-
lator sequences and often involve CTCF. Chromatin hubs (Ins+LCR) allow position-independent
expression of transgenes in mice.
MANCHE TRANSKRIPTIONSFAKTOREN BILDEN HOMODIMERE DURCH INTERAKTION VON ZN-

FINGERN.
a. richtig
b. falsch ?
Eukaryotic transcription factors possess a basic region (cluster of positively charged amino acids)
necessary for DNA binding which are often coupled to a dimerisation motif such as leucine zip-
pers or helix-loop-helix motifs. Zinc fingers are DNA binding domains present in numerous mam-
malian proteins and can either interact with DNA or proteins. Two distinct types of zinc fingers can
14 of 56
be distinguished: the zinc atom is either complexed with two cysteines and two histidines (C2H2
motif) or four cysteines (C4 motif). Steroid (/nuclear) hormone receptors contain a C4 zinc finger
motif and can bind to DNA either as a homodimer (glucocorticoid receptor) or a heterodimer (RXR
family members).
DIE CTD DER RNA POLYMERASE II BINDET TRANSKRIPTIONSAKTIVATOREN.

a. richtig
b. falsch
To allow promotor clearance TFIIH phosphorylates RNA Pol II at serine 5 (Ser5) of its carboxy-
terminal domain (CTD), containing up to 52 heptapeptide-repeats of amino acids which can be
phosphorylated (i.e. Tyr-Ser-Pro-Thr-Ser-Pro-Ser). However, transcription is not yet possible be-
cause of the elongation block. Hence, a second phosphorylation at Ser2 in the heptapeptide-re-
peats of CTD by the elongation factor P-TEFb is needed in order to allow transcriptional elonga-
tion. The C-terminal domain of RNA Pol II is also essential for the interaction with the core of the
mediator complex which mediates between RNA polymerase II and transcription factors, thereby
coordinating transcriptional initiation and elongation. Furthermore, phosphorylation by the tran-
scription factors TFIIH and PTEFb causes association of proteins that participate in mRNA pro-
cessing.
CHROMATIN REMODELLING KANN DIE DNASE I HYPERSENSITIVITÄT VON DNA BEREICHEN ÄN-
DERN.
a. richtig
b. falsch
Open (i.e. transcribed) regions of chromatin can be mapped by their DNase I hypersensitivity. If
chromatin is exposed to low concentrations of DNase I, the enzyme will cleave sequences specif-
ically where nucleosomes either have been removed or altered (DNA is no longer tightly wrapped
around the nucleosome). Furthermore, promotors, enhancers and other transcriptional control re-
gions are hypersensitive to DNase I; the more loosely the DNA is packed, the more accessible it is
to a nuclease such as DNase I.
BEI NIEDRIGEM SAUERSTOFFPARTIALDRUCK LIEGT TF YAP1 REDUZIERT UND INAKTIV VOR.

a. richtig
b. falsch
Molecular oxygen creates an oxidative environment in the cell and nucleus, leading to disulfide
bond formation and activation of the transcription factor YAP1. Thereby the YAP1 molecule is
bent and its nuclear export sequence (NES) is hidden; transcription is active. However, if oxygen
concentrations are low, the YAP1 molecule’s SH groups are reduced and NES is no longer ob-
scured. Therefore, YAP1 is exported from the nucleus, leading to transcriptional arrest.  
15 of 56
TRANSKRIPTIONSFAKTOREN KÖNNEN DIE BINDUNG VON CHROMATIN REMODELLING KOM-
PLEXEN AN DAS CHROMATIN BEWIRKEN.
a. richtig
b. falsch
scription factors (or both). Vice versa, binding of transcription factors can either be cause or con-
sequence of chromatin remodelling and / or chromatin modification.
TF HIF WIRD DURCH NIEDRIGEN SAUERSTOFFPARTIALDRUCK AKTIVIERT.

a. richtig
b. falsch
In presence of molecular oxygen the hypoxia-induced factor (HIF) is ubiquinated and degraded.
The transcription factor HIF regulates the response to low oxygen levels in the cell by activating
genes which will raise oxygen concentrations. As long as enough oxygen present, HIF is pro-
duced, ubiquinated and degraded. This continuous production allows a rapid response to drop-
ping oxygen concentrations. If oxygen levels in the cell are too low, ubiquitination is inhibited and
the HIF molecule is stable, enabling dimerisation with a second HIF molecule. As a dimer HIF can
activate transcription.
ALLE STEROIDHORMONREZEPTOREN BINDEN / ERKENNEN DIE SELBE DNA SEQUENZ.

a. richtig
b. falsch
Binding sites for nuclear hormone receptors form either inverted repeats (palindromes) or direct
repeats. Homodimers of glucocorticoids and sex hormones bind to palindromic sequences,
whereas heterodimeric receptors of the RXR family bind to direct repeats. Those direct repeats
are sequentially always the same but differ in the spacing between them. Thereby nuclear hor-
mone receptors can recognise and bind to each sequence specifically.
PHOSPHORYLIERUNG VON NFΚB REGULIERT DIE INTERAKTION MIT DER INHIBITORISCHEN UN-
TEREINHEIT IΚB.
a. richtig
b. falsch
NFκB consists of two subunits which are complexed with the inhibitory protein IκB. Binding of an
extracellular ligand to a membrane-bound receptor emits a signal stimulating the IκB kinase,
which subsequently phosphorylates IκB. Phosphorylation IκB marks for ubiquitination and hence
for degradation, enabling NFκB to enter the nucleus and stimulate target gene expression. CBP is
a co-activator of NFκB, not only helping to activate target gene promotors but also acetylating
NFκB to increase its activity. Phosphorylation of the histone acetyltransferase CBP increases its
affinity for NFκB and thereby enhances transcriptional activity of the latter.
16 of 56
ALLE STEROIDHORMONREZEPTOREN SIND PERMANENT AN DNA GEBUNDEN.
a. richtig
b. falsch
Some nuclear hormone receptors are constantly bound to DNA, acting as a transcriptional repres-
sor as long as no ligand is bound. Binding of a specific ligand induces a conformational change in
the nuclear hormone receptor, thereby it now acts as a transcriptional activator. Nuclear hormone
receptors associate with steroids, vitamin D or other lipophilic hormones. Inactive nuclear hor-
mone receptors recognise the activating ligand either in the cytosol (e.g. glucocorticoid-receptor)
or in the nucleus bound to DNA (e.g. RXR-heterodimers).
DAS CTCF PROTEIN IST AN DER BILDUNG VON DNA SCHLAUFEN BETEILIGT.
a. richtig
b. falsch
Chromatin hubs are produced by the interaction between locus control regions with insulator se-
quences and often involve CTCF. CTCF molecules not only bind to one another but also to insula-
tor sequences, thereby facilitating the formation of a DNA loop containing transcribed sequences.
GLUCOCORTICOIDE ERHÖHEN DIE MENGE AN IΚB.

a. richtig
b. falsch
Glucocorticoids suppress the NFκB pathway by increasing IκB synthesis and thereby act anti-in-
flammatory.
DIE AKTIVITÄT DES KOAKTIVATORS ß-CATENIN ERFOLGT DURCH INHIBITION SEINES ABBAUS.
a. richtig
b. falsch
The Wnt signalling pathway is important for development and cell growth in adults. Wnt ligands
inhibit ß-catenin proteolysis, allowing it to act as a transcriptional co-activator. By binding of Wnt
to frizzled, the inhibitory complex can no longer bind ß-catenin nor ubiquitinate it for degradation.
Hence, the active pathway of ß-catenin ensues: under active Wnt signalling, ß-catenin can enter
the nucleus, binds to a complex of transcription factors as a co-activator and thereby enable tran-
scription of target genes.
DNA-BINDING REGIONS OF SOME TRANSCRIPTION FACTORS ARE FORMED BY AMPHIPATHIC

HELICES.
a. richtig
b. falsch
Common DNA binding domains of transcription factors are basic regions, zinc fingers, helix-turn-
helix and beta scaffolds. Basic regions comprise various positively charged amino acids mediat-
ing DNA binding. Basic regions are generally associated to a dimerisation motif such as leucine
zippers, amphipathic helices or helix-loop-helix structures. Zinc fingers possess a Zn2+ atom
17 of 56
complexed either with two cysteines and two histidines (C2H2) or four cysteines (C4); they can
bind both DNA and proteins. Helix-turn-helix structures are typical for prokaryotic transcription
factors (e.g. lac / trp repressors) but can also be present in eukaryotic transcription factors. Beta
scaffolds are DBD of transcription factors such as TBP and Stat1, generally binding to the minor
groove of DNA.
PHOSPHORYLIERUNG DES KOAKTIVATOR PGC1ALPHA INHIBIERT DIE AKTIVIERUNG DES TF

FOXO1.
a. richtig
b. falsch
The Akt pathway inhibits activity of the Foxo1 transcription factor through phosphorylation of its
PGC1-α co-activator. Association of insulin to the insulin receptor triggers activity of the PI3 ki-
nase which activates Akt (Protein Kinase B) through phosphorylation. Akt mediates downstream
responses, including cell survival, growth, proliferation and migration, by phosphorylating serine
and threonine residues on a range of intracellular proteins. In case of PGC1-α and Foxo1, phos-
phorylation leads to inactivation of PGC1-α, and export from the nucleus of Foxo1.
HOHE INTRAZELLULÄRE CHOLESTEROLMENGEN FÜHRT ZUR AKTIVIERUNG DES TF SREB.

a. richtig
b. falsch
The transcription factor SREB (sterol response element binding protein) is produced as a mem-
brane-bound “pre-cursor” protein, localised in the endoplasmatic reticulum. SCAP (SREB cleav-
age activating protein) is a cytosolic protein which binds cholesterol and thereby controls intracel-
lular cholesterol levels. If high concentrations of cholesterol are present, SCAP binds cholesterol;.
However, upon a drop of cholesterol levels, SCAP binds SREB and cleaves its N-terminal domain
off. Hereby, SCAP enables SREB to enter the nucleus and activate transcription of genes which
increase cholesterol uptake or cholesterol synthesis.
TRANSCRIPTION STARTS OF TRANSCRIBED GENES ARE FREE OF NUCLEOSOMES.

a. richtig
b. falsch
moved in order for transcription factors to be able to bind to DNA. Chromatin remodelling com-
plexes can assist in chromatin assembly by moving already deposited histone octamers, generat-
ing room for additional disposition. Remodeller complexes (e.g. SWI/SNF, ISWI) have two distinct
functions, either they lead to site exposure or they enable an altered composition of nucleosomes.
Binding sites can be exposed via three different pathways: chromatin remodellers enable reposi-
tioning of the binding site by nucleosomal sliding, nucleosomal ejection (eviction of the nucleoso-
mal complex) or localised unwrapping of the DNA. The elongation factor FACT (facilitator of tran-
scription) displaces the H2A/H2B dimer from the nucleosome, thereby loosens the contact be-
tween DNA and histone, allowing transcription. An altered composition is achieved either by dimer
exchange whereby a H2A/H2B dimer is exchanged with a histone variant (interplay of chaperones
and chromatin remodellers) or dimer ejection (removal of the H2A/H2B dimers). 
18 of 56
TRYPTOPHAN ACTS AS COREPRESSOR OF THE TRP OPERON.
a. richtig
b. falsch
The Trp operon is an anabolic operon leading to the synthesis of tryptophan; its transcriptional
control requires Trp to act as a co-repressor. When cells are deprived of tryptophan, the repressor
cannot bind to the operator sequence, therefore transcription is active. If intracellular tryptophan
concentrations are sufficiently saturated, tryptophan will associate with the repressor as a co-re-
pressor, enabling transcriptional repression by association to the operator.
TRANSCRIPTION FACTORS ARE ABLE TO CAUSE BINDING OF CHROMATIN REMODELLERS TO

CHROMATIN.
a. richtig
b. falsch
scription factors (or both). Vice versa, binding of transcription factors can either be cause or con-
sequence of chromatin remodelling and/or chromatin modification.
A TRANSCRIPTION BUBBLE CONTAINS MRNA-DNA HYBRIDS.

a. richtig
b. falsch
The transcription bubble is the region of the DNA template where the DNA can be unwound for a
short distance and a DNA/RNA hybrid can be produced.
MRNA IS COMPLEMENTARY TO THE CODING STRAND OF DNA.

a. richtig
b. falsch
The (non-coding) template strand is complementary to the mRNA, whereas the (coding) non-tem-
plate strand is identical to the mRNA.
DNA LOOP FORMATION IS IMPORTANT FOR THE INTERACTION BETWEEN RNA POLYMERASE AND
THE INITIATION COMPLEX.
a. richtig
b. falsch
TATA box binding protein (TBP) as well as TBP associated factors (TAFs) are part of the general
transcription factor TFIID; TBP causes DNA bending around the initiation site. TFIID is the first
protein to bind to DNA during the formation of the pre-initiation complex of RNA polymerase II
(RNA Pol II). As one of the few proteins in the pre-initiation complex that binds DNA in a se-
quence-specific manner, it helps position RNA polymerase II over the transcription start site of the
gene. Binding of TFIID to the TATA box in the promoter region of the gene initiates the recruitment
of other factors such as TFIIA, TFIIB, and TFIIF.  
19 of 56
BISULFIDE SEQUENCING DETERMINES THE METHYLATION OF HISTONES.
a. richtig
b. falsch
Sodium bisulfide conversion is a method enabling detection of genomic CpG methylation. CpG
islands are regions of high CpG density that lack CpG methylation and are found at promoters of
most human genes. Long term silencing of the gene can be ensured by methylation of CpG island
regions. For example, genes on the inactive X-chromosome and certain imprinted genes are si-
lenced this way. During bisulfide sequencing sodium bisulfide will convert every unmethylated cy-
tosine into uracil. Amplification of DNA by PCR will result in a modified AU pairing instead of CG.
THE CTCF PROTEIN IS INVOLVED IN THE ISOLATION OF CHROMATIN REGIONS.

a. richtig
b. falsch
scribed. Chromatin hubs are produced by the interaction between locus control regions with insu-
lator sequences and often involve CTCF. CTCF molecules not only bind to one another but also
to insulator sequences, thereby facilitating the formation of a DNA loop containing transcribed
sequences.
VERBIEGEN DER DNA DURCH DAS CAP-PROTEIN ERLAUBT DIE INTERAKTION VON OPERATOREN
DES LAC OPERONS.
a. richtig
b. falsch
Lac, Gal and Ara operons are catabolic operons, encoding enzymes which allow metabolisation of
lactose, galactose and arabinose via glycolysis. The operon is defined by a control region and
coding region. The control region comprises the promoter for lacI, a CAP (catabolite activator pro-
tein) binding site and an operator sequence. The coding region consists of lacZ, lacY and lacA
genes. Glucose is always preferred to lactose, hence transcription of the lac operon is continu-
ously suppressed by lacI. Upon glucose deprivation, an adenylate cycles forms cyclic AMP
(cAMP) from ATP which acts as a starvation signal for CAP. Association of cAMP with CAP en-
ables the complex to bind to the CAP binding site, introducing a bend in the DNA which facilitates
interaction with the α-subunit (CTD) of RNA polymerase. This interaction results in high levels of
transcription. If both glucose and lactose are present in the cell, lactose removes the repressor
lacI but absence of the starvation signal cAMP hinders CAP-DNA binding. Therefore, lac genes
will be expressed at low transcription levels.
In absence of both glucose and lactose, the highest level of transcriptional repression is executed.
By the interaction of lac repressors bound to operon O1 and O3 a DNA loop is formed which is
further stabilised by CAP.
20 of 56
IN BAKTERIELLEN ZWEIKOMPONENTEN SYSTEMEN FUNGIERT EIN SENSORPROTEIN ALS TRAN-
SKRIPTIONSFAKTOR.
a. richtig
b. falsch
A two-component regulatory system serves as a basic stimulus-response coupling mechanism to

allow organisms to sense and respond to changes in many different environmental conditions.
Two-component systems typically consist of a membrane-bound histidine kinase that senses a
specific environmental stimulus and a corresponding response regulator (TF) that mediates the
cellular response, mostly through differential expression of target genes.
Regulators with a DNA-binding effector domain (helix-turn-helix motif) are the most common re-
sponse regulators, and have direct impacts on transcription. They tend to interact with their cog-
nate regulators (receptor kinases) at an N-terminus receiver domain, and contain the DNA-binding
effector towards the C-terminus. Once phosphorylated at the receiver domain, the response regu-
lator dimerises, gains enhanced DNA binding capacity and acts as a transcription factor.
https://en.wikipedia.org/wiki/Two-component_regulatory_system
https://en.wikipedia.org/wiki/Response_regulator
DAS "RUDDER" DER RNA-POLYMERASE WIRD ZUR TRENNUNG DER DNA-STRÄNGE BENÖTIGT.
a. richtig
b. falsch
E. coli’s RNA polymerase comprises four subunits: two α-subunits, a ß-subunit and a ß’-subunit.
The holoenzyme additionally contains a sigma factor which is responsible for RNA polymerase
specificity (i.e. different sigma factors localise RNA Pol to different genes). Double helical DNA en-
ters a long cleft in the surface of the enzyme, and is held in place by a large flexible portion of the
enzyme termed the “clamp”. Within the cleft, the DNA is separated and RNA is paired to it (repli-
cation bubble). A magnesium ion, sitting at the critical point where RNA nucleotides are added to
the primary transcript, is thought to help catalyse the reaction. An internal barrier forces a bend in
the growing DNA-RNA duplex, exposing the RNA end for addition of the incoming nucleotide. A
short protein extension, termed the “rudder," helps to separate RNA from DNA and the two exit
the polymerase along separate paths.
"BACKTRACKING" DER RNA POLYMERASE IST WICHTIG FÜR REVERSE TRANSKRIPTION.

a. richtig
b. falsch
RNA polymerase backtracking is a regulatory mechanism which allows regulatory pauses and ar-
rests, enables termination mechanisms, ensures transcriptional fidelity (proof reading), controls
the elongation rate, couples transcription and translation in bacteria and facilitates co-transcrip-
tional RNA folding and processing. Proof reading performed by RNA polymerase is achieved ei-
ther by pyrophosphorylitic editing, replacing the last nucleotide of the growing mRNA by a py-
rophosphate, or by hydrolytic editing, whereby the RNA Pol “backtracks” a few nucleotides and
removes a piece of mismatched RNA.
21 of 56
DER TRANSKRIPTIONSFAKTOR TFIIH IST EINE RNA-POLYMERASE KINASE.
a. richtig
b. falsch
To allow promotor clearance TFIIH phosphorylates RNA Pol II at serine 5 (Ser5) of its carboxy-
terminal domain (CTD), containing up to 52 heptapeptide-repeats of amino acids which can be
phosphorylated (i.e. Tyr-Ser-Pro-Thr-Ser-Pro-Ser). However, transcription is not yet possible be-
cause of the elongation block. Hence, a second phosphorylation at Ser2 in the heptapeptide-re-
peats of CTD by the elongation factor P-TEFb is needed in order to allow transcriptional elonga-
tion.
DER MEDIATORKOMPLEX BESTEHT AUS BASALEN (GENERELLEN) TRANSKRIPTIONSFAKTOREN.

a. richtig
b. falsch
The mediator is a multiprotein complex that functions as a transcriptional co-activator in all eu-
karyotes, mediating interactions between RNA polymerase II and transcription factors. The media-
tor complex is a crucial component for transcription initiation. Mediators interact with the pre-initi-
ation complexes, composed of RNA Polymerase II and general transcription factors TFIIB, TFIID,
TFIIE, TFIIF, and TFIIH to stabilise and initiate transcription.
https://en.wikipedia.org/wiki/Mediator_(coactivator)#Chromatin_organization
The core of the mediator interacts with RNA Pol II CTD, coordinating transcriptional initiation and
elongation. Mediator complexes vary depending on which transcription factor they associate with;
they can bind either RNA Pol II or TFs first. Its kinase module contains CDK8/CycC, enabling
phosphorylation of target substrates. However, some mediator complexes do not contain a kinase
module; vice versa, kinase modules can function without the core mediator.
HISTONCHAPERONE HALTEN NUKLEOSOMEN STABIL, DAMIT SIE VON DER ELONGIERENDEN

RNA POLYMERASE NICHT ZERSTÖRT WERDEN.
a. richtig
b. falsch
Histone chaperones are broadly defined as a group of proteins that bind histones and regulate
transcription-coupled and transcription-independent nucleosome assembly. In general, histone
chaperones can be classified either as H3/H4 or H2A/H2B chaperones based on their preferential
binding. Some histone chaperones, like FACT, bind both H3/H4 and H2A/H2B.
If a nucleosome is overlapping a transcription start or transcription factor binding sites, the nucle-
osome needs to be moved. This is achieved, inter alia, through the elongation factor FACT (facili-
tator of transcription), which displaces the H2A/H2B dimer from the nucleosome. Thereby the in-
teraction between DNA and nucleosome is loosened, allowing transcription. FACT also acts as a
histone chaperon to reinstall the H2A/H2B dimer.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4004355/
22 of 56
INSULATOR-ELEMENTE TRENNEN EXON UND INTRON SEQUENZEN.
a. richtig
b. falsch
scribed.
An exon is any part of a gene that will encode a part of the final mature RNA produced by the
gene after introns have been removed by RNA splicing. The term exon refers to both the DNA se-
quence within a gene and to the corresponding sequence in RNA transcripts. In RNA splicing, in-
trons are removed and exons are covalently joined to one another as part of generating the ma-
ture messenger RNA. An intron is any nucleotide sequence within a gene that is removed by RNA
splicing during maturation of the final RNA product. The term intron is derived from intragenic re-
gion, i.e. a region inside a gene. Introns refer to both the DNA sequence within a gene and the
corresponding sequence in RNA transcripts.
https://en.wikipedia.org/wiki/Exon, https://en.wikipedia.org/wiki/Intron
Decker: Offene Fragen
WIE NENNT MAN DIE ABGEBILDETE STRUKTUR?
a. Was bedeutet in diesem Zusammenhang LCR und “insulator”?

b. Welche Chromatin-Modifikationen würden Sie in Bereichen von "active genes" bzw. "inactive
genes" erwarten?
scribed. Chromatin hubs are produced by the interaction between locus control regions with in-
sulator sequences and often involve CTCF. CTCF molecules not only bind to one another but also
23 of 56
to insulator sequences, thereby facilitating the formation of a DNA loop containing transcribed
sequences.
Chromatin regions which harbour actively transcribed genes are defined by acetylation, whereas
transcriptionally silenced regions are methylated. Such post-translational modifications are medi-
ated by histone acetyl transferases (HATs) and histone deacetylases (HDACs).
GENE KÖNNEN DURCH EPIGENETISCHE MECHANISMEN REGULIERT WERDEN.

a. Welches Kriterium muss dabei erfüllt sein?
b. Welche Rolle spielen Polycomb-Proteine (PRC1, PRC2 bei Säugern)?
The term epigenetics describes mitotically stable but reversible modifications to DNA or chro-
matin that lead to changes in gene expression in the absence of genetic alterations. Epigenetic
mechanisms are responsible for different gene expression patterns from identical DNA in different
cells and organs. Epigenetic gene control results from the formation of distinct chromatin struc-
tures (eu-/heterochromatin) and DNA methylation.
Euchromatin contains transcribed regions of the genome with nucleosomal DNA that is not in
compact higher order chromatin structures. It contains “unique” (non-repetitive) DNA dispersed
throughout the nucleus. In contrast, heterochromatin contains less or not transcribed genomic
regions in often tightly packed chromatin. Heterochromatin can be constitutive (centromere /
telomere) or induced (X Chromosome inactivation / silencing of mating type loci in Saccha-
romyces). Chromatin modifications and density distinguish different forms of heterochromatin,
however it generally possesses a low content of acetylated histones. Heterochromatic DNA is
highly repetitiv and predominantly located in the periphery of the nucleus. DNA elements such as
locus control regions (LCR) and insulators (Ins) (and other boundary elements) determine the
boundaries and properties of euchromatin and heterochromatin. DNA methylation patterns are
distinct for euchromatin and heterochromatin, respectively: CpG islands are hypo-methylated (i.e.
not methylated at all) in euchromatin containing transcribed genes, whereas heterochromatin is
extensively methylated.
Polycomb homologues found in mammals are responsible for heterochromatin formation and
propagation. Polycomb complexes are recruited to the DNA by specific transcription factors:
transcriptional repressors facilitate polycomb binding, whereas transcriptional activators prevent
binding of polycomb complexes. Euchromatin is heterochromatised by polycomb complexes
through trimethylation of e.g. H3, resulting either in H3K9Me or H3K27Me. Therefore, polycomb
complexes need to possess both reader and writer functions of histone modifications. PRC1 can
bind H3K9Me as well as H3K27Me, recognises the trimethylation and transfers a ubiquitin onto
the residue. Thereby it interferes with transcription as ubiquinated nucleosomes are not tran-
scribed. PRC2 binds to trimethylated H3K27Me, conferring the same modification onto neigh-
bouring residues. Hence it facilitates spreading of heterochromatin which is prevented by LCR
and Ins at the border to euchromatin.
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BESCHREIBEN ODER SKIZZIEREN SIE DIE STRUKTUR EINES NUKLEOSOMS.
a. Welche Komponenten des Nukleosoms sind besonders gut für posttranslationale Modifikatio-
nen zugänglich? Nennen Sie drei dieser Modifikationen.
b. Können Sie in jedem Fall eine klare Aussage darüber machen, wie sich diese Modifikationen
auf Transkription auswirkt?
A nucleosome is a chromatin structure formed by 146bp DNA

winding approximately twice around a histone octamer compris-
ing two H2A/H2B dimers and a H3/H4 tetramer. Histone H1 as-
sociates with linker DNA between the nucleosomes and causes
compaction of nucleosomal DNA. H2 and H4 possess long N-
terminal unstructured regions (histone tails) protruding from the
nucleosome which are accessible for post-translational modifi-
cations. Acetyl groups attached by histone acetyl transferases
(HATs) neutralise the positive charge of lysines, thereby loosening
the DNA-nucleosome interaction and increasing the accessibility
of DNA for transcription factors. Histone deacetylases (HDACs) exert the opposite effect by re-
moving acetyl groups from lysines in histone tails. Other post-translational modifications include
methylation of arginine or lysine and phosphorylation of threonine or serine. In contrast to acetyla-
tion, the functions of phosphorylation and methylation is not always definitive as the interplay with
other modifications of the histone code determine their effect.
WAS WIRD HIER GEZEIGT?

a. Was ist seine Funktion?
b. Nachweis von seiner (TFIID/Protein) Bindung an DNA in vitro.
Depicted is the general transcription factor TFIID which plays a

central role in the initiation of RNA polymerase II dependent tran-
scription by initiating pre-initiation complex (PIC) assembly at the
core promoter. TFIID comprises the TATA-binding protein (TBP) and
13 TBP-associated factors (TAFs), which specifically interact with a
variety of core promoter DNA sequences.
The first step in the assembly of the initiation complex is usually the
binding of TFIID to the TATA box of the promoter. TBP causes DNA
to bend around the initiation site and the remaining transcription
factors comprising the initiation complex, such as TFIIB, TFIIF, TFI-
IA, TFIID and TFIIH, are recruited.
Sequential formation of the initiation complex from general transcrip-

tion factors can be investigated by the electrophoretic mobility shift assay (EMSA). This method
allows determination of TF binding order by incubating radioactively labelled DNA fragments car-
rying a promoter with various transcription factors. Binding of the proteins to the promoter can be
monitored by electrophoresis on a polyacrylamid gel. With addition of each component a larger
complex was formed resulting in a shift on the gel.
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WARUM MUSS DNA WÄHREND DER TRANSKRIPTION RELAXIERT WERDEN?
a. Nennen Sie beide Enzyme, die an der Relaxion beteiligt sind.
b. Erklären Sie Unterschiede in ihren Mechanismen.
Transcription of topologically constrained DNA templates causes superhelical stress. If a DNA

strand which has been fixated on either end would be transcribed by a RNA polymerase unable to
rotate, this would result in negative supercoiling upstream of RNA Pol and positive supercoiling
downstream of RNA Pol. Ultimately, transcription would arrest.
In relaxed DNA, strands “twist” once around each other every 10.4 bp; however, if a strain is put
on this DNA twists their length either increases or decreases. Twists can be relaxed to form a
toroidal structure which is especially important for eukaryotic nucleosomes. To restore original
twists, “writhe” can be induced (i.e. supercoiling). The linkage number determines how often the
strands / helices are wrapped around each other, either by twist or writhe (linkage number = twist
+ writhe).
Topoisomerases are enzymes which relieve tension of topologically constrained DNA: Topoiso-
merase I induces relaxation of negative supercoils. No ATP is required as they only nick one
strand of DNA resulting in intermediate single-strand breaks through which the other strand is
passed. In contrast, Topoisomerase II facilitates ATP-dependent intermediate double-strand
breaks leading either to relaxation of DNA or establishment of negative supercoils.
SKIZZIEREN SIE DEN SIGNALTRANSDUKTIONSWEG MIT G-COUPLED RECEPTORS UND CREB. WIE
KÖNNEN SIE IN-VITRO UND IN-VIVO NACHWEISEN, DASS CREB AN DIE DNA BINDET?
CREB is a conditionally active, signal-dependent, cell membrane

receptor-dependent resident nuclear factor. This means that CREB
requires activation trough external factors which result in phospho-
rylation of the transcription factor by second messenger signalling
cascades. CREB resides in the nucleus irrespective of its activation
and possesses a basic region for DNA binding.
Some membrane bound receptors use G-proteins to activate adeny-

late cyclase (AC) and generate intracellular cAMP. In prokaryotes,
cAMP functions as an important starvation signal which activates
transcription factors stimulating catabolic operons. In mammals,
cAMP acts not only a metabolic regulator but also an important sig-
nal transducing molecule which has acquired various functions.
cAMP is synthesised in presence of G-protein coupled receptors,
i.e. receptors associated with GTPases, which are activated by GTP
hydrolysis. Active G-proteins stimulate the adenylate cyclase which
produces cAMP from ATP. Intracellular cAMP is then bound by ki-
nase A comprising four subunits: two regulatory subunits and two
catalytic subunits. The regulatory subunits control kinase A activity:
if cAMP is bound, the regulatory subunits dissociate and protein kinase A is activated. The tran-
scription factor CREB is bound to CRE (cyclic AMP response element) but remains inactive as
long as it is not phosphorylated. Phosphorylation of CREB by kinase A enables binding of its co-
activator CBP (a histone acetylase) and subsequent transcription of genes which should be active
when the cell produces cAMP.
The interaction of CBP with phosphorylated CREB has been characterised in detail by using a flu-
orescence polarisation binding assay and a genetic interaction assay in yeast. https://mcb.asm.org/
content/20/5/1546
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A central tenet of the CREB–CBP model is that CREB binds constitutively to the CRE and that
regulation occurs through the phosphorylation-dependent recruitment of CBP. Immunoprecipita-
tion assays show that CREB does not interact in vivo with CRE, or similar elements in several
other genes. Rather, CREB binding in vivo is regulated in a cell-specific manner, a finding that was
confirmed by using in vivo genomic footprinting assays. https://www.pnas.org/content/101/37/13572
Note: I could not find anything in my notes or on the slides on in vivo or in vitro detection mechanisms. Hopefully one of
the abovementioned assays is correct.
WAS BEWIRKT GENOMIC IMPRINTING VON GENEN?

a. Welche Genmodifikation ist mit genomic imprinting assoziiert?
b. Die Bindung welches TF wird durch diese Modifikation reguliert?
In diploid organisms some loci of the maternal and paternal haploid genomes carry particular
markers (i.e. are imprinted) which distinguish them. Imprinting causes differential expression of
maternal and paternal imprinted genes through silencing, which is essential for the development
and homeostasis of mammalian organisms. Imprinting needs to be erased and reestablished in
embryonic gonads preceding sex determination.
There are three different models of genomic imprinting. First, CTCF binds to the imprinting control
region (ICR) on the maternal chromosome enabling transcription of a downstream promoter while
simultaneously repressing upstream gene expression. Methylation of the paternal chromosome
ICR prevents CTCF from binding. Thereby upstream gene expression is now possible, while ex-
pression of downstream genes is repressed. Second, CTCF binds to the non-methylated ICR on
the maternal chromosome and recruits an associated protein which induces formation of hete-
rochromatin. Hence, CTCF insulates downstream active transcription sites from the upstream
heterochromatic sequences. On the paternal chromosome, ICR is methylated enabling heter-
chromatisation of downstream sequences. Third, both maternal and paternal chromosome con-
tain a sequence for a long non-coding RNA (Air). On the maternal chromosome Air is not ex-
pressed, allowing transcription of upstream promoters. However, on the paternal chromosome, Air
is expressed which not only leads to promoter occlusion on neighbouring promoters but also to
inhibition of gene expression of other upstream promoters by recruitment of HATs. Thereby, ge-
netic imprinting ensures that only one copy of each gene is active. 
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SKIZZIEREN SIE DIE STRUKTUR EINES NUKLEOSOMS EINSCHLIEßLICH DNA. BENENNEN SIE DIE
KOMPONENTEN.
a. Welchen Änderungen ist nukleosomale DNA im Zuge des chromatin remodelling unterworfen?
b. Wie bzw. durch welche Proteine wird die Energie für das chromatin remodelling erzeugt?
If the transcription start or transcription factor binding sites

are overlapped by nucleosomes, the nucleosomes have to be
moved in order for transcription to be possible. Chromatin
remodelling is achieved by ATP-dependent chromatin remod-
eller complexes such as SWI/SNF or ISWI. Two distinct
mechanisms define the function of chromatin remodellers:
site exposure and altered composition. Site exposure hap-
pens when chromatin remodelling results in repositioning or
ejection of the nucleosome, or in unwrapping of the DNA from
the nucleosome. Dimer exchange, introducing histone vari-
ants to the nucleosome, and dimer ejection are both mecha-
nisms propagating altered composition of the nucleosome. The latter is achieved, inter alia, by the
elongation factor FACT (facilitator of transcription) which not only displaces the H2A/H2B dimer
from the nucleosome, allowing transcription, but also reinstalles the dimer by acting as a histone
chaperone.
NENNEN SIE READER, WRITER UND ERASER DES HISTONE CODES.
The histone code comprises covalent modifications which are transduced by the histone-modify-
ing writer enzymes and removed by the antagonising activity of erasers. Writers are classified into
families according to the type of modification they confer: histone acetyl transferases (HATs)
acetylate lysin residues of protruding histone tails; kinases phosphorylate serine and threonine;
protein arginine methyltransferases (PRMTs) methylate arginine residues; histone lysine methyl-
transferases (HKMT) confer methylations onto lysine. Those particular histone tail modifications
are recognised by protein domains termed readers: bromodomains recognise acetylations;
chromodomains recognise methylated lysine; Tudor recognises methylated arginine; whereas
phosphorylations are recognised by yet unknown complexes. However, writer enzymes do not
solely confer modifications trough their catalytic subunit but also possess a reader function
through their bromo- or chromodomain. Erasers of the histone code are classified by their enzy-
matic function as well: histone deacetylases (HDACs) remove acetyl groups from lysine; protein
phosphatases (PPTases) remove phosphorylations from serine and threonine residues; deaminas-
es remove methylations from arginine and amine oxidases and hydroxylases remove methylations
from lysine residues on the histone tail.
WAS IST DER UNTERSCHIED ZWISCHEN DE NOVO METHYLIERUNG UND MAINTENANCE

METHYLIERUNG? WIE IST EINE DNA TEMPLATE FÜR DE NOVO METHYLIERUNG BESCHAFFEN
UND WIE EINE TEMPLATE FÜR MAINTENANCE METHYLIERUNG?
Methylation of CpG dinucleotides is achieved either “de novo” or through “maintenance”: de novo
methylation by DNA methyltransferases Dnmt3a and Dnmt3b is required if a DNA strand does not
possess any prior methylation (e.g. during embryogenesis). After semi-conservative DNA replica-
tion a progeny strand is hemimethylated and paired with a methylated parental strand, depending
on the maintenance of methylation by Dnmt1 to restore symmetry. 
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GENE KÖNNEN DURCH EPIGENETISCHE MECHANISMEN REGULIERT WERDEN. BENENNEN SIE
DAS UNTEN DARGESTELLTE EPIGENETISCHE PHÄNOMEN.
a. Wofür steht die Abkürzung ICR und welches Protein bindet an die ICR?
b. Welche epigenetischen Modifikationen reguliert die Bindung des Proteins an die ICR und
welche Wirkung hat die Modifikation auf seine Bindung?
Genomic imprinting depends on the so-called imprinting control region (ICR), a sequence
present on both maternal and paternal chromosomes, which enables controlled, selective expres-
sion of upstream and downstream promoters on each chromosome. CTCF can only bind to ICR if
it is non-methylated which results in inhibition of upstream gene expression while downstream
promoters can be transcribed. When ICR is methylated, CTCF cannot bind and upstream gene
expression is ensured. Furthermore, binding of CTCF regulates distribution and formation of hete-
rochromatin: if CTCF is bound, upstream sequences will heterochromatise while downstream
gene expression active through insulation by CTCF. However, if ICR is methylated and CTCF bind-
ing prevented, downstream sequences will become heterochromatin, allowing expression of up-
stream promoters.
WELCHE DNA BASE KANN IN EUKARYOTISCHEN ORGANISMEN METHYLIERT VORLIEGEN?

a. Skizzieren Sie die Struktur.
b. Welche biologische Funktion hat die Methylierung von DNA?
DNA methylation is mostly (but not exclusively) associated with transcriptional

silencing. Most cytosine bases within a CpG dinucleotide are methylated at the
C5 atom in vertebrate genomes. An exception to this rule are the so-called CpG
islands which are often found at the 5’ end of house keeping genes (i.e. genes
that are constitutively active). DNA methylation not only contributes to epige-
netic silencing (X Chromosome inactivation, imprinted genes) but also to the
stable silencing of endogenous retroviruses, retrotransposons and transgenes.
Methylation of CpG dinucleotides is achieved either “de novo” or through “maintenance”: de novo
methylation by DNA methyltransferases Dnmt3a and Dnmt3b is required if a DNA strand does not
possess any prior methylation (e.g. during embryogenesis). After semi-conservative DNA replica-
tion a progeny strand is hemimethylated and paired with a methylated parental strand, depending
on the maintenance of methylation by Dnmt1 to restore symmetry.
29 of 56
Long term silencing of genes can be achieved by methylation of CpG islands whose methylation
then can be recognised by MeCP that silences chromatin by recruiting HDACs and methyltrans-
ferase complexes. Thereby, MeCP proteins remove any markers indicating active transcription.
POLYCOMB COMPLEXES ARE IMPORTANT FOR EPIGENETIC GENE REGULATION.

a. What is their effect on gene expression?
b. How do they produce this effect?
c. Which enzymatic activity of polycomb complexes is important?
Polycomb homologues found in mammals (PC) are responsible for heterochromatin formation
and propagation. Polycomb complexes are recruited to the DNA by specific transcription factors:
transcriptional repressors facilitate polycomb binding, whereas transcriptional activators prevent
binding through polycomb complexes. Euchromatin is heterochromatised by polycomb complex-
es through trimethylation of e.g. H3, resulting either in H3K9Me or H3K27Me. Hence, polycomb
complexes need to possess both reader and writer functions of/for histone modifications. PRC1
can bind H3K9Me as well as H3K27Me, it recognises the trimethylation and transfers a ubiquitin
onto the residue. Thereby it interferes with transcription as ubiquinated nucleosomes are not tran-
scribed. PRC2 binds to trimethylated H3K27Me, conferring the same modification (methylations)
onto neighbouring residues. Thereby it facilitates spreading of heterochromatin which is prevented
by LCR and Ins at the border to euchromatin.
Polycomb homologues found in Drosophila (PcG), together with trithorax complexes (Trx) are re-
sponsible for epigenetic control of cellular (transcriptional) memory. Both Trx and PcG families in-
clude proteins that covalently modify histones and proteins that non-covalently modify chromatin;
covalent modifications on histones can increase binding by non-covalent modifying complexes.
Active genes in Drosophila euchromatin are marked by transcriptional activators which recruit
trithorax complexes to antagonise polycomb complexes. Thereby, trithorax complexes maintain
heritable state of active gene expression. Transcriptional repressors recruit polycomb complexes
which enable heritable genetic silencing.
SIE HABEN MITTELS DNA-MICROARRAY NACHGEWIESEN, DASS DIE ZUGABE VON EINEM WACH-
STUMSFAKTOR DIE EXPRESSION VON 120 GENEN ERHÖHT.
a. Wie können Sie nachweisen, dass tatsächlich Transkriptionskontrolle vorliegt?
b. Sie vermuten, dass der Transkriptionsfaktor Stat3 mit den identifizierten Genen wechselwirkt
bzw. diese stimuliert. Wie weisen Sie das nach?
DNA microarrays (/ gene chips) allow genome-wide analysis of gene expression, i.e. analysis of
the transcriptome. Varying colours of fluorochromes associated to specific genes depict different
level of transcription. Microarray data can be displayed as heat maps and clusters of co-regulated
genes: hierarchical clustering associates genes into a group if they have a similar behaviour to a
certain environmental stimulus. Clusters of co-regulated genes allow the definition of cell type-
specific transcriptomes. They can be used, inter alia, to determine the origin of metastatic tumour
cells. Transcriptional control can be determined by Gro-Seq (genomic run on sequencing) which
is an advancement of the nuclear run on assay. This method allows simultaneous analysis of tran-
scription of various genes and even transcriptome analysis (i.e. genome wide analysis of nascent
transcripts). Nuclei are isolated and transcripts are elongated in presence of chemically modified
UTP. Subsequently, nascent, chromatin-associated mRNA is isolated by antigen binding of modi-
fied U and transcribed into cDNA, which is then sequenced. Bioinformatic analysis of results en-
ables evaluation of data, subsequently providing information on transcript frequencies. Tthe more
30 of 56
a certain part of a gene has been transcribed, the higher the sequence reads of this particular
gene and its transcription.
To determine whether or not Stat3 influences expression of a certain gene under a certain condi-
tion, a reporter gene assay can be implemented. A cell is co-transfected with two plasmids, the
first carrying the gene encoding the transcription factor Stat3, the second a reporter gene and a
Stat3 binding site. If Stat3 stimulates transcription upon environmental stimulation, more reporter
gene transcripts will be synthesised as Stat3 is more active. However, genetic proof showing the
relevance of a particular transcription factor is achieved by loss-of-function mutations. TF function
can be impaired either by siRNA mediated knock-down or knock-out (i.e. gene targeting through
homologous recombination or CRISPR). Gene expression patterns of isolates of a Stat3 wild type
and a Stat3 knock-down mutant can be compared by Gro-Seq or microarray analysis.
DIE RNA POLYMERASE II BESITZT C-TERMINALE DOMÄNE.

a. Wie ist diese beschaffen?
b. Welche Rolle spielen Phosphorylierungsereignisse im Transkriptionszyklus?
RNA Pol II possesses a carboxy-terminal domain (CTD) comprising up to 52 heptapeptide-repeats

of amino acids which can be phosphorylated (i.e. Tyr-Ser-Pro-Thr-Ser-Pro-Ser). During transcrip-
tional initiation, the general transcription factor TFIIH serves two purposes: unwinding the DNA at
the transcription start site (via its 3’→5’ helicase and 5’→3’ helicase activity) and phosphorylating
serine 5 (Ser5) of CTD. The initiation complex containing various TFs and RNA Pol II is formed at
the core promotor, however promotor clearance is not yet possible until a second phosphorylation
event. The second phosphorylation of CTD at Ser2 is conferred by the elongation factor PTEFb
which thereby not only enables transcriptional initiation but also lifts the elongation block. The C-
terminal domain of RNA Pol II is also essential for the interaction with the core of the mediator
complex which mediates between RNA polymerase II and transcription factors, thereby coordi-
nating transcriptional initiation and elongation. The RNA Polymerase II transcription cycle is de-
pendent on phosphorylation and dephosphorylation of the CTD, as its phosphorylation by TFIIH
and P-TEFb facilitates not only transcriptional elongation but also the association of proteins that
participate in mRNA processing.
DNASE I WIRD ZUM KARTIEREN VON HYPERSENSITIVEN BEREICHEN VERWENDET.

a. Was ist Hypersensitivität, wie funktioniert sie?
b. Welche regulatorischen DNA Elemente sind hypersensitiv?
Open (i.e. transcribed) regions of chromatin can be mapped by their DNase I hypersensitivity. If
chromatin is exposed to low concentrations of DNase I, the enzyme will cleave sequences specif-
ically where nucleosome have either been removed or altered (DNA is no longer tightly wound
around the nucleosome). Furthermore, promotors, enhancers and other transcriptional control re-
gions are hypersensitive to DNase I; the more loosely the DNA is packed, the more accessible it is
to a nuclease such as DNase I.
To determine DNase I hypersensitivity, chromatin has to be isolated and digested with different
concentrations of DNase I; partially digested DNA is purified by removing proteins. Subsequently,
purified DNA is cut by restriction enzymes and separated on a Southern Blot applying probes for
the gene of interest. Specific band patterns indicate hypersensitive regions: undigested chromatin
forms thick bands at the top of the blot whereas lightly digested chromatin forms bands nearer
the middle of the blot. However, heavily digested and therefore hypersensitive chromatin forms no
fragment at all.  
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WAS SIND DIE SPEZIELLEN FUNKTIONEN VON TFIIH-KOMPLEXEN?
The general transcription factor TFIIH has three distinct functions depending on its state: the core
complex comprises two helicases which unwind DNA both in 3’→5’ direction and 5’→3’ direction.
One TFIIH holoenzyme, complexed with a CTD kinase, is responsible for promoter clearance
through phosphorylation of RNA Pol II at Ser5 of its CTD. Whereas the other TFIIH holoenzyme is
essential in DNA repair through core-associated DNA repair proteins. If RNA Pol II encounters a
“problem” during elongation, TFIIH is recruited and associated repair proteins target the particular
sequence.
SKIZZIEREN SIE DEN INITIATIONSKOMPLEX BEI DER TRANSKRIPTION DURCH RNA POL II.
WELCHE DIESER PROTEINE ASSOZIIEREN MIT DNA-BINDENDEN FAKTOREN?
TFIID is the first protein to bind to DNA during the formation of the pre-
initiation complex of RNA polymerase II. As one of the few proteins in the
pre-initiation complex that binds DNA in a sequence-specific manner, it
helps position RNA polymerase II over the transcription start site of the
gene. TFIID contains the TATA box binding protein (TBP) as well as TBP
associated factors (TAFs); TBP causes DNA bending around the initiation
site. Binding of TFIID to the TATA box in the promoter region of the gene initiates the recruitment
of other transcription factors such as TFIIA (stabilises the interaction of TBP and DNA), TFIIB
(links TBP to RNA Pol II), TFIIF, TFIIE (enables docking of TFIIH) and TFIIH (phosphorylates CTD).
Transcription by RNA polymerase II also depends on the mediator complex which not only as-
sociates with the CTD of RNA Pol II during the formation of the pre-initiation complex, but also
binds recruited transcription factors and DNA. Thereby, it can mediate between the different com-
ponents of the initiation complex, and ultimately enable transcriptional elongation. Furthermore,
TFIIB possesses not only a linker function but also a reader function. The linker binds the rudder
of RNA polymerase II, aiding strand separation and subsequent open complex formation (tran-
scription bubble), whereas the reader helps recognise the transcription start and to position RNA
Pol II precisely.
WELCHE ZWEI TYPEN DER STEROIDHORMON REZEPTOREN KENNEN SIE?

a. Wo befinden sie sich?
b. Wo und wie binden sie an DNA?
Steroid (/nuclear) hormone receptors associate with steroids such as glucocorticoids and sex
hormones as well as with vitamin D or other lipophilic hormones (retinoic acid / thyroid hormone).
Due to their lipophilic nature, hormone receptors do not require cell membrane bound surface re-
ceptors. Some steroid hormone receptors may reside in the cytoplasm and enter the nucleus
upon ligand binding (glucocorticoid receptor), while other (nuclear) receptors such as RXR family
members reside inside the nucleus, constantly bound to DNA. The latter constitutively repress
transcription until a ligand is bound and the receptors undergo a conformational change enabling
their function as transcriptional activators.
Homodimeric hormone receptors such as glucocorticoid and oestrogen receptors bind inverted
repeats (palindromes) of DNA. Heterodimeric hormone receptors such as TR/RXR and RAR/RXR
(retinoid X receptor) recognise the same direct repeats. However, receptors can bind specifically
to their target sequence as binding motifs can be distinguished through the spacing between the
direct repeats. 
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METHODEN UM MRNA QUANTITATIV NACHZUWEISEN. WELCHE LIEFERN EXAKTE QUANTITATIVE
WERTE? KANN MAN DAMIT TRANSKRIPTION NACHWEISEN?
Northern Blots depict radioactively visualised band patterns which represent mRNA molecules
separated by their respective size. Prior, RNA is isolated from cells and separated on agarose
gels, subsequently mRNA is blotted onto a nitrocellulose membrane and labelled with a radioac-
tive probe specific for the target mRNA (cDNA of target gene). Autoradiography enables detection
of RNA/DNA hybrids.
RT-PCR synthesises cDNA from mRNA isolates through reverse transcription with help of primers
specific for a certain gene. Amounts of cDNA synthesised can be visualised on an agarose gel.
RT-PCR is a semiquantitative method as only the first 30 cycles lead to duplicating amplification,
thereafter primers are saturated and amplification is no longer linear.
qPCR enables real-time observation of cDNA amplification as fluorophores intercalate into dsDNA
fragments. The intensity of fluorescence is proportional to the amount of synthesised DNA, allow-
ing determination and evaluation of the linear, quantitative phase of PCR. Moreover, cDNA con-
centrations obtained are proportional to the amount of mRNA which had been present in the cell.
Gene microarrays can only be applied if the whole genome of a target organism is known, as
complementary DNA sequences of each gene are fixed in defined positions onto a glass chip.
mRNA isolates from a organism treated with different environmental stimuli are reverse tran-
scribed into fluorescent-labelled cDNA, which hybridises with its complementary sequence on the
chip. Thereby it is possible to determine activity of genes in a particular environment, since each
cDNA has its specifically coloured probe allowing comparative analysis.
NGS (next generation sequencing) is a method for gene expression analysis whereby mRNA is
reverse transcribed into cDNA. It is based on automated massive parallel sequencing of cDNA
(libraries) to render sequence reads which are complementary to mRNA isolates. Bioinformatics
determine transcript frequencies for individual genes enabling quantification of mRNA in the cell.
WELCHE POSTTRANSLATIONALEN MODIFIKATIONEN VON TRANSKRIPTIONSFAKTOREN KENNEN

SIE? NENNEN SIE EIN BEISPIEL.
Cells react to environmental stimuli by changes in gene expression, which are achieved either by
de novo synthesis or posttranslational modification of transcription factors. There are five distinct
ways a transcription factor can be activated: binding of a ligand such as heavy metals, protein
modifications such as phosphorylations, cleavage of a precursor molecule, dissociation of an
inhibitory molecule. The transcription factor ACE1 is a sensor for excess Cu2+ and induces genes
involved in heavy metal detoxification. At low levels of Cu2+ ACE1 remains inactive. However, if
Cu2+ concentrations increase, ACE1 binds Cu2+ and associates with its binding site on DNA. This
induces transcription of Metallothionine, whose gene product binds intracellular metals like cop-
per.
SKIZZIEREN SIE DIE EREIGNISSE DIE VON DER BINDUNG EINES WACHSTUMSFAKTORS (EGF) AN
SEINEN REZEPTOR ZUR INDUKTION VON GENEXPRESSION FÜHREN. BENENNEN SIE DIE KOM-
PONENTEN UND RELEVANTE MODIFIKATIONEN DIE AN DER SIGNALTRANSDUKTION BETEILIGT
SIND.
The MAPK signalling pathway is induced by various growth factors (hormones, polypeptides)
receptors. When a growth factor binds to the extracellular domain of a RTK, its dimerisation is
triggered with other adjacent RTKs. Dimerisation leads to a rapid activation of the receptor’s cyto-
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plasmic kinase domains, the first substrate for these domains being the receptor itself. The acti-
vated receptor becomes autophosphorylated on multiple specific intracellular tyrosine residues.
https://en.wikipedia.org/wiki/Receptor_tyrosine_kinase#Epidermal_growth_factor_receptor_family
Association of EGF with a receptor tyrosine kinase (RTK) first results in autophosphorylation, fol-
lowed by the phosphorylation of the GTPase Ras, which then can activate the kinase RAF. RAF
activates MEK, which in turn activates MAPK, which is now able to activate p90RISK and enter the
nucleus. Inside the nucleus MAPK phosphorylates TCF (turnally complex factor), while SRF
(serum response factor) is phosphorylated by p90RISK. Phosphorylated TCF and SRF can bind to
SRE regions, which are present on many genes stimulated by MAPK signalling pathways (e.g. the
oncogene c-fos).
WELCHE ENZYMATISCHEN AKTIVITÄTEN VON ZELLOBERFLÄCHENREZEPTOREN KENNEN SIE?

WIE SIND SIGNALTRANSDUKTIONSWEGE BESCHAFFEN, DIE DIESE ENZYMATISCHE AKTIVITÄT
LETZTLICH IN VERÄNDERTE GENEXPRESSION ÜBERTRAGEN + JE 1 BSP.
Major enzymatic activities of membrane bound cell surface receptors are phosphorylation through
kinase activities, proteolysis and hydrolysis.
Cytokine receptors can be coupled with JAK kinases which phosphorylate tyrosines of the tran-
scription factor STAT, enabling its dimerisation and binding to target genes. Cytokine TGFß binds
a receptor which phosphorylates serine and threonine residues of R-Smad, which can enter the
nucleus after it has bound a “common” SMAD, where the complex activates transcription.
Receptor tyrosine kinases are specific for growth factors such as hormones or polypeptides,
and binding results in induction of MAPK signalling pathways. The receptor phosphorylates itself,
thereby increasing its own activity, and then leads to activation of various kinases including RAS,
RAF, MEK and MAPK (in this order). Phosphorylated MAPK is able to enter the nucleus and stimu-
late expression of target genes.
G protein-coupled receptors are receptors coupled to GTPases which by GTP hydrolysis stimu-
late cAMP production by adenylate cyclases. cAMP then binds to the regulatory subunit of protein
kinase A (PKA), allowing dissociation of the catalytic subunits and kinase activity. CREB bound to
CRE (cyclo-AMP response element) is activated through phosphorylation by PKA and transcrip-
tion of CRE is initiated.
Delta-Notch receptor interaction causes cleavage of Notch receptor C-terminus that acts as a
transcriptional regulator. Notch is responsible for lateral inhibition of cells adjacent to epithelial
cells which will become sensory neurons, preventing those neighbouring cells from becoming
neurons too. Interaction between Delta and Notch happens directly through cell-cell contact. If
34 of 56
Delta associates to Notch, the C-terminal domain of Notch is cleaved off by a protease activity.
The Notch tail enters the nucleus where it binds to CSL, stimulating target gene expression.
WIE VIELE RNA-POLYMERASEN GIBT ES IN EUKARYOTEN?

a. Was sind ihre Produkte?
b. Wie sehen ihre typischen Promotorregionen aus?
RNA Polymerase I is localised in the nucleolus

and responsible for rRNA transcription; its core
promoter (comprising pppA or pppG?) is preceded
by an upstream regulatory sequence (URS) and
followed by a downstream terminator sequence.
The transcription start resides within the core
promoter. RNA Polymerase II is localised in the
nucleoplasma where it transcribes pre-mRNAs
and snRNAs (small nuclear). Protein coding genes
possess an upstream enhancer sequence, fol-
lowed by URS and the TATA box motif, as well as
a CAP binding site followed by an A or G nu-
cleotide?. The transcription start is adjacent to the
latter. snRNA promoter sequences comprise an
upstream enhancer and a CAP binding site fol-
lowed by an A nucleotide? after which the tran-
scription start ensues. RNA Polymerase III is localised in the nucleoplasma as well, transcribing
tRNAs, 5S rRNAs, snRNAs and ncRNAs (non-coding). The promoter sequence of tRNAs con-
sists of URS (containing pppG?) followed by the transcription start which is enclosed between
URS and two binding? regions A and B; downstream sequences comprise poly-TA. Promoter se-
quences for 5S rRNA are similar, the only difference being that the transcription start is enclosed
by URS (comprising pppG?) and binding sites A and C; its downstream sequence also possesses
poly-TA. snRNA promoter sequences are similar to the promoter sequence of RNA Pol II: an en-
hancer precedes the TATA motif followed by pppG and the transcription start; downstream are
poly-TA nucleotides localised.
Note: I am not sure if I interpreted the depiction correctly; hence the question marks.
ERKLÄREN SIE DIE BEDEUTUNG VON SIGMA-FAKTOREN. WIE WÜRDEN SIE NACHWEISEN, DASS
EIN GEN VON SIGMA 70 REGULIERT WIRD?
Depending on the presence or absence of a sigma factor, the RNA polymerase is defined as a
holoenzyme and a core enzyme, respectively. The holoenzyme binds DNA nonspecifically and
performs a one-dimensional search for the promoter. After a specific promoter is found and RNA
Pol has associated, a closed complex is formed, followed by a conformational transition to an
open complex. This allows promoter clearance, dissociation of the sigma factor and transcription-
al elongation by the core enzyme. Different sigma factors confer particular RNA Pol specificities
and thereby allow transcription of various genes. Prominent examples of sigma factors are sigma
70, responsible for housekeeping genes; sigma 32, essential to the transcription of heat shock
genes; sigma 28, specific for genes involved in mobility and chemotaxis.
35 of 56
To determine whether transcription is possible due to sigma 70, DNA sequences containing the
gene of interest and RNA polymerase have to be isolated. Both are then incubated under three
different conditions: once with the target factor sigma 70, once with another sigma factor and
once without any sigma factor. If indeed sigma 70 is solely responsible for transcription of certain
genes, no reads (e.g. on a Northern Blot) should be visible under the other two conditions.
NENNEN SIE 2 CHROMATIN-REMODELLIERENDE KOMPLEXE.

a. Beschreiben Sie die biochemischen Mechanismen von Chromatin Remodellers.
b. Woraus beziehen sie die dafür nötige Energie?
Chromatin remodelling complexes are not only able to

bind to histones but also possess an ATPase domain
which converts ATP to ADP. Three mechanisms are ap-
plied by chromatin remodellers, either leading to site ex-
posure or altered composition. Nucleosomal sliding re-
sults in the repositioning of a transcription factor binding
site, whereas rotational repositioning leads to unwrap-
ping of the nucleosome (both site exposure mechanisms).
However, nucleosomal transfer can lead to multiple out-
comes: either a nucleosome is ejected (site exposure),
H2A/H2B dimers are exchanged or ejected (altered com-
position). ATP-dependent chromatin remodelling com-
plexes are, inter alia: SWI/SNF possessing a Bromod-
omain for histone binding and ISWI comprising three different domains for histone binding, such
as a Bromodomain, a SANT domain and a PHD finger.
The ATPase of SWI/SNF (human BAF complex) is bound to DNA and moves in respect to the
Bromodomain (translocation). This translocation induces stress which is released through nucleo-
somal remodelling: stable products include nucleosomal sliding, DNA bulging, histone exchange
and nucleosomal transfer. ISWI facilitates nucleosomal sliding by binding to the H4 tail protruding
from the nucleosome and inducing the so-called HSS-out state (Hand-Sant-Slide domain; primed
state). ATP hydrolysis results in the HSS-in conformation, whereby HSS is moved in regard to the
ATPase and DNA is translocated 7 bp per hydrolysed ATP.
ERKLÄREN SIE, WIE VON DER INITIATIONSSTELLE DER TRANSSKRIPTION ENTFERNT BINDENDE
TRANSKRIPTIONSFAKTOREN DIE BINDUNG DER RNA POLYMERASE BEEINFLUSSEN KÖNNEN.
Transcription factors partaking in transcriptional initiation either bind directly to the promoter se-
quence or to distal enhancers. To ensure communication between transcription factors and RNA
polymerases, an enhanceosome has to be formed. This structure requires association of high
mobility proteins, bending the DNA so that interaction of transcription factors is possible. For in-
stance, the IFN-ß enhanceosome mediates binding of histone modifying and chromatin remodel-
ling complexes to the IFN-ß promoter, as a prerequisite for the removal of the nucleosome at the
transcription start and initiation complex formation.
Another way to enable long range interactions is mediated through the transcription factor CTCF,
which is very often involved in the formation of chromatin hubs. Chromatin hubs are structures
induced through the interaction of insulator sequences with each other or with CTCF, thereby
forming a loop which brings the locus control region (LCR) into close proximity with the target
genes. Interaction of LCR with promoter or enhancer sequences determines which genes within a
36 of 56
chromosomal domain are transcribed. Moreover, chromatin hubs not only decrease distances be-
tween sequences whose interaction is essential for gene expression, they can also separate ac-
tively transcribed regions from non-transcribed regions.
Moll: Altfragen
WAS IST IF3 UND WIE KANN MAN SEINE FUNKTION IN VITRO BZW. IN VIVO FESTSTELLEN?
SKIZZE.
Translation initiation factor IF3 is one of the three factors (IF 1-3) required for the initiation of pro-
tein biosynthesis in bacteria. IF3 is thought to function as a fidelity factor during the assembly of
the pre-initiation complex which consists of the 30S ribosomal subunit, the initiator tRNA and
mRNA. IF3 is a basic protein that binds to the 30S ribosomal subunit, controlling tRNA associa-
tion by reducing the affinity of the fMet-tRNAfMet and discriminating against non-cognate initiation
complexes (i.e. non-AUG start codons) trough conformational changes (i.e. stronger binding to
the codon). Due to the localisation of the C-domain of IF3 at the interface side of the platform of
the ribosomal 30S subunit, the factor sterically blocks subunit joining. After release of IF3, a dy-
namic conformational switch, introduced by binding of GTP to IF2 stimulates the rate of 50S and
30S subunit joining (to form the 70S ribosome).
Toeprinting is a technique that was developed in the translation field to measure precisely the
position of ribosomes (or ribosomal subunits) on known mRNAs. It relies on the fact that a reverse
transcriptase is stopped when it encounters a blocking complex (ribonucleoprotein or protein) on
the RNA. The length of the primer extension product produced when the RNA is occupied by
such a complex compared to the full-length primer extension product indicates the 3' position of
the complex of interest. https://www.ncbi.nlm.nih.gov/pubmed/24003204
To determine IF3 function in vitro, a known mRNA sequence containing a ribosomal binding site
(RBS) is incubated with radioactively labelled primers (designed to bind downstream of RBS) and
nucleotides as well as fMet-tRNA, once with IF3 and once without IF3. If IF3 indeed is essential
for correct positioning and pre-initiation complex formation (30S, IF1, IF2 and IF3), reverse tran-
scription will arrest at the pre-initiation complex at position +15 in samples containing IF3. There-
by a shorter cDNA fragment is obtained (toeprint signal) than from reverse transcription of sam-
ples without IF3 (extension signal). Sequence fragments are then separated by gel electrophoresis
and visualised by radioactively labelled probes on a Southern Blot.
To determine IF3 activity in vivo, a reporter gene assay can be implemented. For this purpose, a
plasmid encoding the reporter gene lacZ with the start codon AUG is introduced into cell contain-
ing wild type IF3; ß-galactosidase can be synthesised. If cells containing mutant IF3 are trans-
duced with the same plasmid production of ß-galactosidase is possible as well. However, if a
plasmid containing lacZ and an AUU start codon is introduced into cells possessing wild type IF3
37 of 56
ß-galactosidase cannot be synthesised. IF3 undergoes a conformational change when bound to a
start codon other than AUG which results in stronger binding preventing recruitment of the 50S
subunit and subsequent transcription (IF3 discriminates against other codons). When the same
plasmid is introduced into cells possessing a mutant version of IF3, ß-galactosidase can still be
produced but only with a third of the original translational efficiency. Mutant IF3 is no longer able
to undergo the same conformational change which allows wild type IF3 to discriminate between
AUG and other start codons. Therefore, it does not exhibit increased binding affinity to AUU,
rather it initiates translation of ß-galactosidase.
Note: Answers on in vitro and in vivo detection mechanisms are adapted from Kleinotti.
WIE KANN MAN EXPERIMENTELL FESTSTELLEN, DASS EIN TF DIE SHINE DALGARNO SEQUENZ
BINDET? NENNEN SIE ZWEI EXPERIMENTE. SKIZZE!
Prokaryotes possess “internal initiation” which means that their translation initiation site is defined
by the direct interaction of the ribosome with the initiation region (/ribosomal binding site). This
interaction is achieved through rRNA / mRNA association of anti-Shine-Dalgarno (aSD) and Shine-
Dalgarno (SD) sequences, respectively. The Shine-Dalgarno SD sequence is a ribosomal binding
site in bacterial mRNA, generally located around 8 bases upstream of the start codon AUG. SD
sequence helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning the ri-
bosome with the start codon. The length of aSD and SD sequences bound to each other and their
corresponding strength of interaction define the stability of ribosomal binding. If sequences are
too long and binding is too stable, the ribosome is not able to move; hence translation is not pos-
sible.
Initiation of prokaryotic translation depends on the formation of the initiation complex at the SD
sequence, comprising the initiator tRNA (fMet-tRNA), IF1, IF2 and IF3 as well as the small 30S ri-
bosomal subunit associated with S1. In E. coli translational initiation S1 is essential as it is re-
quired for translation of mRNAs with canonical ribosomal binding sites and a 5’ UTR (untranslated
region). It binds 30S ribosome by direct protein-protein interactions and interacts with an en-
hancer sequence upstream of SD as part of the translation initiation complex.
Note: Neither do I understand whether she really wanted to know how transcription factors would bind the Shine-Dal-
garno sequence (TF are essential to transcription not translation) nor which experiments she would like to have listed as
an answer. Guessing she meant initiation factors I would answer again with toeprinting and maybe immunoprecipitation.
WIE KANN DAS BINDEN VON RIBOSOME AN DER RIBOSOMENBINDUNGSSTELLE VERHINDERT

WERDEN? WIE KANN MAN DIESE MECHANISMEN DETEKTIEREN?
In prokaryotes transcriptional and translational control relies on riboswitches, which form upon
receiving stimuli from some external event usually associated with the metabolic status of the cell.
Those stem loop structures either terminate transcription prematurely or sequester the Shine–Dal-
garno sequence and thereby inhibit translation initiation. RNA involved in stem loop structure for-
mation can be divided into two domains: the binding domain or aptamer, and the regulatory do-
main containing the ribosomal binding site (RBS). The aptamer has a structured binding pocket
expressing high affinity and high specificity for ligands; the regulatory domain reacts with a struc-
tural change upon ligand binding by the aptamer and thereby alters gene expression. Transcrip-
tional control is mediated by the mutually exclusive formation of an anti-anti-terminator stem or an
intrinsic anti-terminator stem. Translational control is mediated by the mutually exclusive formation
of an anti-anti-RBS stem or an anti-RBS stem.
38 of 56
mRNA can engage various secondary structures
such as inhibitory stem loop structures which can
mask ribosomal binding sites, and thereby effective-
ly inhibit translation initiation. So-called “breathing”
allows mRNA to obtain a single-stranded conforma-
tion enabling binding by the ribosome and subse-
quent transcription. For instance, phage λ possess-
es two translation initiation sites which encode two
different gene products: a lethal hole-former (holin),
essential to the lytic pathway and an inhibitor of
hole-formation enabling lysogeny. The Shine-Dal-
garno sequence of the hole-former is masked by a
stem loop structure, therefore translation of holin is
dependent on a conformational change induced by
binding of S1 to a poly-U sequence. Binding results
in a ss-conformation of mRNA by abolishing the in-
hibitory stem loop structures allowing induction of
holin.
Thermosensors (or RNA thermometers, RNAT) are

structural elements located within the 5’ UTR of protein-coding genes and control its translation
by operating as reversible molecular “zippers” or “switches" that unmask the RBS in response to
temperature changes. For instance, E. coli exhibits both forms of conformational change: a heat
shock induces increased translation of sigma 32 through “unzipping” of structures which previ-
ously masked the start codon. A cold shock results in a conformational “switch”, refolding the
mRNA in such a way that the Shine-Dalgarno sequence becomes more accessible and the cold
shock protein A (CspA) can be translated.
Riboswitch structures can be detected by in-line probing: RNAs are incubated either with a cer-
tain metabolite or without the metabolite and sequence reads are compared. The dominant path-
way by which RNA degrades involves an “in-line” nucleophilic attack by the 2′ oxygen on the ad-
jacent phosphorus centre. Cleavage only takes place if the attacking 2′ oxygen, the phosphate
and the departing 5′ oxygen of the phosphodiester bond approach a linear configuration (“in-
line”). The speed at which spontaneous cleavage occurs depends on the local structural context
in which each RNA linkage exists. Linkages that reside in highly structured regions of a folded
RNA (for example, a base-paired helix) typically resist cleavage because the relevant atoms are
not held in a linear configuration.
39 of 56
WAS IST RIBOREGULATION? UNTERSCHIED CIS/TRANS ENCODED SRNAS MIT ZWEI BEISPIELEN
ERKLÄREN.
The term riboregulation is used when RNA acts as a genetic regulator influencing translation effi-
ciency. Riboregulators are non-coding, antisense RNAs, i.e. single-stranded RNAs that are com-
plementary to a protein coding mRNA with which they hybridises, hence blocking its translation
into protein. https://en.wikipedia.org/wiki/Antisense_RNA
Riboregulators can be categorised into two distinct groups: cis-encoded RNA and trans-encoded
RNA. The former leads to transcriptional attenuation, translational inhibition, inhibition of the for-
mation of an activating pseudoknot and promotion/inhibition of mRNA degradation; the latter is
responsible for mediating bacterial responses to environmental stresses.
Cis-encoded RNAs are encoded by the same genetic locus as their target gene and have the po-
tential to form perfect RNA duplexes. Inhibition of primer maturation by the cis-encoded anti-
sense RNA RNAI: a mechanism which has only been found for ColE1 and its related plasmids,
requiring a plasmid encoded replication primer that is synthesised as a 550 nts pre-primer by
RNAII. The formation of a persistent RNAII/DNA hybrid is dependent on specific secondary and
tertiary structures of RNAII which form during RNAII synthesis. Subsequently, the mature primer
which can be extended by DNA polymerase I, is generated through cleavage of the RNA strand of
the RNAII/DNA hybrid by RNase H. The binding of the antisense RNA (RNAI) induces a change in
the nascent primer, thereby preventing primer maturation. The kissing complex between RNAI and
RNAII is stabilised by the plasmid-encoded Rom protein.
Trans-encoded RNAs are encoded on a locus distinct from the target site (i.e. are not genetically
linked), resulting in the formation of imperfect RNA duplexes. Thereby trans-encoded RNAs are
able to regulate multiple mRNAs and affect multiple targets via alternative base pairing. Trans-en-
coded RNAs require the additional factor HFQ, a heat-stable, hexameric protein which mediates
the interaction between ncRNAs and targets by acting as a RNA chaperone. HFQ interacts with
many small regulatory RNAs and mRNAs, affecting their stability by introducing conformational
changes in both sRNAs and mRNAs. Moreover, it acts as a pleiotropic regulator affecting quorum
sensing, biofilm formation and bacterial growth. An important feature of HFQ is its association-
dependent protection of sRNAs from degradation by RNase E. RyhB is a trans-encoded small
RNA which is essential in iron homeostasis and can induce translational silencing. If free iron is
present in the cell, the ribosome binds to the RBS of sodB, enabling translation of proteins need-
ed in the presence of iron or requiring iron for their function. In the absence of intracellular iron,
RyhB is transcribed and binds to sodB. HFQ, stimulating the interaction between RyhB and the
translation initiation region, recruits RNase E which degrades both sRNA and mRNA whereby fur-
ther translation is prevented. Furthermore, trans-encoded sRNAs can induce translational activity
(e.g. DsrA activates rpoS).
VERGLEICHEN SIE CIS-ANTISENSE-SRNA AUS BAKTERIEN MIT SI-RNA IN EUKARYOTEN! SKIZZE!
The term riboregulation is used when RNA acts as a genetic regulator influencing translation effi-
ciency. Riboregulators are non-coding, antisense RNAs, i.e. single-stranded RNAs that are com-
plementary to a protein coding mRNA with which they hybridises, hence blocking its translation
into protein. https://en.wikipedia.org/wiki/Antisense_RNA
Riboregulators can be categorised into two distinct groups: cis-encoded RNA and trans-encod-
ed RNA. The former leads to transcriptional attenuation, translational inhibition, inhibition of the
formation of an activating pseudoknot and promotion/inhibition of mRNA degradation; the latter is
responsible for mediating bacterial responses to environmental stresses.
40 of 56
There are various transcripts which can adopt dsRNA structures that can be processed by Dicer
into siRNAs (small interfering). Those transcripts include repeat-associated transcripts (cen-
tromeres, telomeres), viral RNAs, hairpin RNAs, convergent transcripts or other anti-sense pairs,
gene/pseudogene duplexes, transgene transcripts and Tasi RNAs. Duplexes can be intra- or in-
termolecular, and although most are perfectly base paired, some are not (e.g. hairpin RNAs and
gene/pseudogene duplexes). A siRNA consists of a guide strand which assembles into functional
siRISC, and a passenger strand which is ejected and degraded. All forms of siRISC contain the
siRNA bound to an Ago protein and usually also some additional factors. Target RNAs are recog-
nised by base pairing and silencing ensues through one of several mechanisms. In many species,
the siRNA populations that engage a target can be amplified by the action of RNA-dependent
RNA polymerase (RdRP) enzymes, strengthening and perpetuating the silencing response.
During canonical RNA interference (RNAi), siRISC recognises a perfectly complementary mRNA,
leading to Ago-catalysed mRNA cleavage at a single site within the duplex. After cleavage, func-
tional siRISC is regenerated, whereas the mRNA fragments are further degraded. siRISC is also
capable of recognising targets with partial complementarity, resulting in translational inhibition
similar to miRNA-like mechanisms involving translational repression and exonucleolytic degrada-
tion. Futhermore, siRISC can induce heterochromatin formation by associating with nascent tran-
scripts and RNA polymerases. In plants, target engagement leads to the association or activation
of a DNA methyltransferase (DMT) that methylates the DNA. In S. pombe and presumably in ani-
mals too, the pathway involves a histone methyltransferase (HMT) that methylates Lys9 of histone
H3, thereby inducing heterochromatinisation. In most eukaryotes other than insects and mam-
mals, target recognition by siRISC induces the synthesis of secondary dsRNAs and siRNAs by
RdRP enzymes. The secondary dsRNAs are processed by Dicer into siRNAs, which add to the
pool of siRISC. In nematodes, many of the secondary siRNAs arise as single-stranded, unprimed
transcripts with 50-triphosphates and do not require Dicer processing.  
41 of 56
WIE KÖNNEN RNA-MOLEKÜLE IN DER TRANSLATION AUF TRANSLATIONS-BEEINFLUSSENDE
PROTEINE EINFLUSS NEHMEN? SKIZZEN
a. Wie kann man das Binden nachweisen?
b. Wie kann man nachweisen, dass es genau das protein of interest ist, dass an das RNA
Molekül gebunden ist?
RNA chemical probing uses chemicals that react with unpaired

bases of RNAs such as CMTC modifying uracil, DMS modifying
adenine and cytosine, or kethoxal which modifies guanine. Their
reactivity depends on local RNA structure, i.e. base-pairing or
accessibility, meaning that differences in reactivity can serve as
a footprint of structure along the sequence. However, target po-
sitions on the RNA can also be protected from the reagents by
binding proteins, enabling application of chemical probing to
assay protein-binding. In chemical probing, structured RNA is
reacted with probing reagents which form a covalent adduct on
the RNA at the site of reaction. When RNA is reverse tran-
scribed into a DNA copy by a reverse transcriptase, the cDNA
generated is truncated at modified positions because the en-
zyme is blocked by the adducts. cDNA sequences of various
truncated lengths therefore inform on the frequency of reaction
at every base position, which in turn reflects the structure profile
along the RNA. cDNA sequences are usually separated and vi-
sualised by gel electrophoresis; the intensity of bands inform
the frequency of observing a truncation at a certain position.
Recent approaches use high-throughput sequencing to achieve
a greater throughput and sensitivity.
https://en.wikipedia.org/wiki/Nucleic_acid_structure_determination
To determine association of a protein to mRNA, mRNA is incu-
bated with the protein and one of the abovementioned reagents
which modify unpaired bases. Thereafter, the extract is purified and the mRNA denatured. Subse-
quently the mRNA is reverse transcribed using labelled primers, binding various positions on the
mRNA, and radioactively labelled nucleotides. cDNA synthesis arrests at modified bases generat-
ing various cDNA fragments of different lengths which can be separated by gel electrophoresis
and visualised on a Southern Blot. In comparison to a cDNA fragments obtained by reverse tran-
scription of mRNA which had not been provided with the particular protein, band patterns should
differ, resembling structural changes in the riboswitch.
Immunoprecipitation can determine where and how a protein binds: mRNA fragments are incu-
bated with the metabolite and are cross-linked by UV-radiation. If the target molecule is bound,
42 of 56
the RNA-protein complex can be isolated by immunoprecipitation through metabolite-specific an-
tibodies. During purification of the metabolite-antibody complex cross-linked mRNA is isolated as
well allowing identification of the specific mRNA sequence bound by RNA sequencing.
Note: After studying both my notes and the lecture slides, I still couldn’t find any answer to this question. But IF she
somehow tried to insinuate that she wants to hear something about riboswitches, I would answer as above.
Bläsi: Altfragen
UNTERSCHIEDE VON SIRNA UND MIRNA IN EUKARYOTEN NENNEN UND SKIZZIEREN. CHARAK-
TERISIEREN SIE IHRE AUSWIRKUNG AUF GENEXPRESSION.
There are various transcripts which can adopt dsRNA structures that can be processed by Dicer
into siRNAs (small interfering). Those transcripts include repeat-associated transcripts (cen-
tromeres, telomeres), viral RNAs, hairpin RNAs, convergent transcripts or other anti-sense pairs,
gene/pseudogene duplexes, transgene transcripts and Tasi RNAs. Duplexes can be intra- or in-
termolecular, and although most are perfectly base paired, some are not (e.g. hairpin RNAs and
gene/pseudogene duplexes). A siRNA consists of a guide strand which assembles into functional
siRISC, and a passenger strand which is ejected and degraded. All forms of siRISC contain the
siRNA bound to an Ago protein and usually also some additional factors. Target RNAs are recog-
nised by base pairing and silencing ensues through one of several mechanisms. In many species,
the siRNA populations that engage a target can be amplified by the action of RNA-dependent
RNA polymerase (RdRP) enzymes, strengthening and perpetuating the silencing response.
During canonical RNA interference (RNAi), siRISC recognises a perfectly complementary mRNA,
leading to Ago-catalysed mRNA cleavage at a single site within the duplex. After cleavage, func-
tional siRISC is regenerated, whereas the mRNA fragments are further degraded. siRISC is also
capable of recognising targets with partial complementarity, resulting in translational inhibition
43 of 56
similar to miRNA-like mechanisms involving translational repression and exonucleolytic degrada-
tion. Futhermore, siRISC can induce heterochromatin formation by associating with nascent tran-
scripts and RNA polymerases. In plants, target engagement leads to the association or activation
of a DNA methyltransferase (DMT) that methylates the DNA. In S. pombe and presumably in ani-
mals too, the pathway involves a histone methyltransferase (HMT) that methylates Lys9 of histone
H3, thereby inducing heterochromatinisation. In most eukaryotes other than insects and mam-
mals, target recognition by siRISC induces the synthesis of secondary dsRNAs and siRNAs by
RdRP enzymes. The secondary dsRNAs are processed by Dicer into siRNAs, which add to the
pool of siRISC. In nematodes, many of the secondary siRNAs arise as single-stranded, unprimed
transcripts with 50-triphosphates and do not require Dicer processing.
A miRNA (micro) is a small non-coding RNA molecule that functions in RNA silencing and post-
transcriptional regulation of gene expression. miRNAs target mRNAs by base-pairing with com-
plementary sequences; as a result, these mRNA molecules are silenced, either by mRNA degra-
dation (perfect complementary) or by translational inhibition (partial complementary). miRNA
genes are usually transcribed by RNA polymerase II; the resulting transcript is capped with a spe-
cially modified nucleotide at the 5' end, polyadenylated (poly-A tail) and forms a hairpin structure
termed pri-miRNA. pri-miRNA is cleaved into pre-mRNA by Drosha and exported into the cyto-
plasm where it is spliced again by Dicer removing the loop of the hair pin. The mature, single-
stranded miRNA is then integrated into the miRISC complex called miRNP (miRNA ribonucleopro-
tein complex). Nonrepressed (i.e. translated) mRNAs recruit initiation factors and ribosomal sub-
units and form circularised structures that enhance translation. However, when miRISCs bind to
mRNAs, they can repress initiation at the cap recognition stage by competing for cap binding or
at the 60S recruitment stage. Alternatively, they can induce deadenylation of the mRNA and
thereby inhibit circularisation of the mRNA. They can also repress a post-initiation stage of trans-
lation by inducing ribosomes to drop off prematurely or promote mRNA degradation by inducing
deadenylation followed by decapping.
TL;DR: siRNAs and miRNAs both regulate gene expression yet they differ in their origination: siRNAs originates from
dsRNA whereas miRNAs originates from ssRNA that forms hairpin secondary structures. siRNA most commonly re-
sponds to foreign RNA (usually viral) and is often perfectly complementary to its target sequence. In contrast, miRNA is
generally not perfectly complementary to its target sequence and regulates post-translational gene expression.
44 of 56
WAS SIND DIE GEMEINSAMKEITEN UND UNTERSCHIEDE VON KANONISCHER UND IRES INITIA-
TION?
In contrast to prokaryotic ribosomal recruitment depending on rRNA-mRNA interactions, eukary-

otic translational initiation relies on protein-protein interactions, as is the case in Cap-dependent
and Cap–independent translation initiation via IRES (internal ribosomal entry site).
To initiate cap-dependent (canonic) translation the CAP structure has to be accessible for the
40S ribosomal subunit. Upon binding of 40S, the mRNA is scanned in an ATP-dependent manner
either for a start codon or a Kozak sequence (favouring initiation complex formation). In cap-de-
pendent translation the cap-binding complex eIF4F comprising eIF4E, eIF4A and eIF4G binds via
eIF4E to the 5′ end of the mRNA (m7G). The capped 5′- end is then associated with 40S complex
by the bridging protein eIF4G which is also bound to eIF4A and the RNA helicase unwinding 5′
secondary structures. The Poly-A binding protein (PABP) binds the poly (A) tail and brings 5′-end
and 3′-end of the mRNA together through the interaction with eIF4G. The pre-initiation complex
results in 5’→3’ scanning of the mRNA and subsequent initiation of codon recognition and hy-
drolysis of eIF2-bound GTP. Thereafter, 40S and 60S ribosomal subunits are joined, forming the
80S eukaryotic ribosome, which allows translation.
Cap-independent translational initiation is facilitated by IRES, an eukaryotic mRNA sequence that

allows the ribosome to initiate polypeptide translation without migrating from the 5’ end. IRES was
first discovered in picornavirus which possesses the viral protease 2A, cleaving eIF4G and there-
by disrupting the eIF4F cap-binding complex. The highly structured 5 ́ noncoding region and lack
of a cap at the 5 ́ end of viral mRNA prevent cap binding and ribosome scanning. Instead, transla-
tion is mediated by IRES, which recruits ITAFs (IRES-transacting factors) and the 40S ribosome
subunit, resulting in the pre-initiation complex formation. The ribosome then recognises the au-
thentic start codon and initiates translation.
Note: Shown is the general structure of an eukaryotic mRNA illustrating some post-transcriptional regulatory elements
for gene expression and their activity. 5′UTR mediated regulation may involve: the 7-methyl-guanosine cap, hairpin-like
secondary structures, RNA-protein interactions, upstream ORFs and internal ribosome entry sites (IRES). 3′UTR mediat-
ed regulation may involve: antisense RNA interactions, RNA-protein interactions involving also multiprotein complexes,
cytoplasmic polyadenylation elements (CPE) and a poly-A tail of varying size.
45 of 56
GEGENÜBERSTELLUNG DES MRNA ABBAUS IN PROKARYOTEN UND EUKARYOTEN. WIE KÖNNEN
SIE NACHWEISEN, OB EINE RNA VOM 5' ODER VOM 3' ENDE AUSGEHEND ABGEBAUT WIRD?
In eukaryotes, most mRNAs undergo decay via the deadenylation-dependent pathway: The 3’
poly-A tail is removed by a deadenylase activity. Following deadenylation, two mechanisms can
degrade the mRNA: either decapping followed by 5′→3′ decay or 3′→5′ decay mediated by the
exosome. In the decapping pathway, a ring complex associates with the 3′ end of the mRNA
transcript and induces decapping of the 5’ end. This leaves the mRNA susceptible to decay by
the 5′→3′ exoribonuclease XRN1. Alternatively, the deadenylated mRNA can be degraded in the
3′→5′ direction by the exosome, with the remaining cap structure being hydrolysed by the scav-
enger-decapping enzyme. Endonuclease-mediated mRNA decay is initiated by the internal
cleavage of the mRNA, which generates two fragments with one unprotected end each. The
fragments are degraded either by the 5′→3′ exoribonuclease XRN1 or in 3′→5′ direction by the
exosome. In S. cerevisiae, deadenylation-independent pathways require recruitment of the de-
capping machinery, whereby the enhancer of decapping-3 (Edc3) is stimulated to engage the de-
capping enzyme. Following decapping, the mRNA is degraded by XRN1 in 5’→3’ direction.
In prokaryotes mRNA decay is a form of gene expression regulation and achieved by RNAses
either belonging to endoribonucleases (RNase E), 3’ exoribonucleases (PNPase) or 5’ exoribonu-
cleases (RNase J). Exoribonucleases degrade mRNA by cleaving single nucleotides off, whereas
endonucleases cleave mRNA inside its sequence resulting in fragments of multiple nucleotides in
length. The 5’ dependent pathway for initiating mRNA degradation engages a PNPase which
converts the 5’ terminal triphosphate of the primary transcript into a monophosphate. The full-
length decay intermediate is then degraded either by a 5’ exoribonuclease or an endonuclease.
Polyadenylation assisted RNA decay in E. coli leads to addition of poly-A tails by Poly-A poly-
merase I (PAP I) to the 3’ ends of an RNA substrate which then provides the single stranded bind-
ing site for both PNPase and RNase II that initiate the degradation. While PNPase catalyses both
3’→5’ phosphorolytic degradation in presence of inorganic phosphate and 5’→3’ polymerisation
in presence of NDPs, RNase II can only degrade RNA hydrolytically in 3’→5’ direction. Both ri-
bonucleases pause upon encountering a G/C rich secondary structure. PNPase either dissociates
relatively quickly or reverses its activity to polymerise polynucleotide tails. Dissociation of PNPase
may initiate multiple rounds of polymerisation by PAP I. In contrast, RNase II remains bound to the
base of the secondary structure thereby effectively blocking the binding of either PAP I or PN-
Pase.
Note: I couldn’t find any answer on detection mechanisms for 3’ or 5’ mediated decay.
RIBOSWITCH AUFBAU ERKLÄREN MIT ZEICHNUNG. WIE KANN MAN FEEDBACK-HEMMUNG EX-
PERIMENTELL NACHWEISEN?
46 of 56
intrinsic anti-terminator stem. Translational control is
mediated by the mutually exclusive formation of an
anti-anti-RBS stem or an anti-RBS stem.
Transcriptional attenuation by the metI riboswitch

containing a SAM binding motif could be confirmed
by in vitro transcription. The motif binding to S-
adenosylmethionine is a highly conserved aptamer
present in riboswitches regulating genes involved in
sulfur metabolism across a broad spectrum of bac-
terial species. Furthermore, SAM-dependent ri-
boswitches regulate expression of genes essential
for survival and/or virulence in medically important
pathogens. Transcription is stimulated by an anti-
terminator stem loop structure, no transcriptional
termination sequence is present on metI. However,
termination of transcription happens in a rho-inde-
pendent manner through transcription of a poly-U
sequence which leads to destabilisation of the in-
teractions between RNA, DNA and polymerase.
Formation of an anti-anti-terminator stem loop in-
hibits transcription. Transcriptional analysis could
proof transcriptional control by the metI riboswitch
by incubating DNA template, RNA polymerase and
nucleotides together; once with target metabolites and once without. Templates without the me-
tabolite will render long (anti-terminator) fragments, however if metabolites are provided, anti-anti-
terminator stem loops are induced resulting in short fragments.
Expression of the cobalamin (coenzyme B12) cob operon in Salmonella typhimurium is controlled
by feedback inhibition: high concentrations of the end-product result in repression protein syn-
thesis. This regulation is conferred mainly at the translational level and involves a cobalamin-in-
duced folding of an RNA hairpin (coenzyme B12 riboswitch). Normally, the translational en-
hancer activates translation by long-distance interaction with the stem of the RBS hairpin. How-
ever, excessive coenzyme B12 binds the B12 box which results in blocking of the translational
enhancer and in a conformational change that sequesters the ribosomal binding site of the cob
mRNA. Thereby binding of the ribosome and subsequent translation initiation is prevented.
Note: I couldn’t find anything on detecting feedback inhibition but chemical probing and in vitro transcription (as for
metI) could work. The presentation of the slightly similar mechanism of autoregulation of ribosome synthesis lacked
the introduction of detection methods as well.
Ribosomal protein genes are organised in operons which often contain genes for both small and large subunit proteins.
Some non-ribosomal proteins that have roles in translation process also encoded in these operons. Genes located in
ribosomal operons either act as a regulator, encoding rRNA binding proteins, or are controlled by translational feedback
regulation through the regulator. For each operon there is a regulatory sequence in the ribosomal protein operon that is
similar to the sequence on the rRNA. Autoregulation of translational balances ribosomal protein levels with rRNA synthe-
sis; i.e. the pseudoknot of S15-mRNA can control gene expression by interacting with the ribosome and inducing the
entrapment mechanism of repression. If S15 is overproduced in the cell, the protein binds and stabilises the pseudoknot
in its own mRNA. The S15-mRNA complex is then loaded onto the ribosome and establishes SD/anti-SD interactions
with the 16S rRNA. However, the ribosome cannot melt the pseudoknot structure so that the initiation codon cannot
reach the ribosome P site to interact with the initiator tRNA. As a consequence, the 3′ end of the mRNA rests on the
surface of the ribosome rather than inside the mRNA channel of the ribosome. The resulting ‘entrapped’ complex is
stalled in the inactive pre-initiation state. In the absence of S15, the rpsO mRNA either unfolds on the ribosome or binds
the ribosome in the unfolded conformation so that the mRNA enters the mRNA channel in the single-stranded confor-
47 of 56
mation. In this position, the initiation codon can be placed into the P site and interact with the initiator tRNA, thereby
initiating translation of the mRNA.
DEFINIEREN SIE DEN BEGRIFF MOLECULAR MIMIKRY. INWIEFERN SPIELT MOLECULAR MIMIKRY
WÄHREND DER TRANSLATION EINE ROLLE? (SKIZZEN)
Molecular mimicry are structural, functional or immunological similarities

shared between macromolecules found on infectious pathogens and in
host tissues. Molecular mimicry plays an important role in immune re-
sponses to infection and in autoimmune diseases. There are three types
of mimicry: the first type of molecular mimicry is identical amino acid
sequences present in different protein molecules; the second type of
molecular mimicry is due to structural similarities rather than amino acid
sequence identities in the mimicking chemical structures; the third type
of molecular mimicry is the recognition of completely dissimilar chemical
structures on separate molecules by a single antibody.
With regard to translation, molecular mimicry can enable translation, lead

to translational arrest, recycling of the ribosome or trans-translation. For
instance, during translational elongation, elongation factor EF-Tu (GTPase)
is recruited to the ribosome and delivers aminoacyl-tRNA to the A site of
the ribosome. Codon/anti-codon interaction triggers a conformational
change which leads to GTP hydrolysis and dissociation of EF-Tu from the
ribosome. EF-G is a molecular mimic of the ternary complex formed by
EF-Tu, GTP and aminoacyl-tRNA, which binds in the vicinity of the A site.
Thereby EF-G pushes the tRNA with the attached nascent polypeptide
from the A site to the P site. The “empty” tRNA which was previously
bound to the P site is shifted into the E site and a new aminoacyl-tRNA
can be bound in the A site. If puromycin is bound by the ribosome in-
stead of an aminoacyl-tRNA, the nascent peptide is transferred onto
puromycin, terminating protein synthesis. Moreover, if the ribosome re-
lease factor (RRF) and EF-G are bound by the ribosomal A site, GTP hydrol-
ysis leads (via yet unknown steps) to dissociation of ribo-
somal subunits releasing all still bound factors and the
mRNA; ribosomal subunits now can be recycled. Trans-
translation by the tmRNA-SmpB complex relies on molecu-
lar mimicry of tRNA, whereby the ternary tmRNA-SmpB
complex is bound in the A site of the ribosome. Subse-
quently the associated alanine residue is decoded as a tag
and integrated into the nascent peptide; the tagged
polypeptide is then degraded by proteolysis. 
48 of 56
WIE KÖNNEN SIE MIT 2 VERSCHIEDENEN METHODEN NACHWEISEN, DASS EIN TRANSLA-
TIONALER REPRESSION IM BEREICH DER SD-SEQUENZ AN EINER PROKARYONTISCHEN MRNA
BINDET? SKIZZEN.
Toeprinting is a technique that measures

precisely the position of ribosomes or pro-
teins on known mRNAs. It relies on the
fact that a reverse transcriptase is
stopped when it encounters a blocking
complex (ribonucleoprotein or protein) on
the RNA. The length of the primer exten-
sion product produced when the RNA is
occupied by such a complex compared to the full-length primer extension product indicates the
3' position of the complex of interest. https://www.ncbi.nlm.nih.gov/pubmed/24003204
To determine if a translational repressor binds to the Shine-Dalgarno sequence, a known mRNA

sequence containing a Shine-Dalgarno sequence is incubated with radioactively labelled primers
(designed to bind downstream of SD sequence) and radioactive-labelled nucleotides. Two condi-
tions are tested: once a repressor thought to bind to the Shine-Dalgarno sequence is added to the
reaction and once the mRNA is incubated solely with nucleotides and the primer. If the repressor
indeed binds to the SD sequence, reverse transcription will arrest at the position of the Shine-Dal-
garno sequence. Thereby a shorter cDNA fragment is
obtained (toeprint signal) than from reverse transcription
of samples without the repressor (extension signal). Se-
quence fragments are then separated by gel elec-
trophoresis and visualised by radioactively labelled
probes on a Southern Blot.
Co-immunoprecipitation can determine where and

how a protein binds: mRNA fragments containing a
Shine-Dalgarno binding site are incubated with the re-
pressor suspected of SD-binding. The repressor and
mRNA are cross-linked by UV-radiation and the repres-
sor is immunoprecipitated by repressor-specific anti-
bodies. During purification of the repressor-antibody
complex cross-linked mRNA is isolated as well allowing
identification of the specific mRNA sequence bound by
RNA sequencing. Thereby it can be determined whether
the repressor is indeed bound to the Shine-Dalgarno
sequence.
HOW CAN A RIBOSWITCH FUNCTION AS A TRANSLATIONAL REPRESSOR IN BACTERIA (EXAMPLE,

DRAWING)? HOW CAN YOU SHOW EXPERIMENTALLY THAT A LIGAND BINDS TO THE APTAMER
DOMAIN OF THE RIBOSWITCH AND THAT IT THEREBY INHIBITS TRANSLATION (2 TECHNIQUES,
DRAWINGS)!
Thiamine pyrophosphate (TTP or cocarboxylase) functions as a coenzyme for decarboxylase en-

zymes such as pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, transketolase; thus it is
a key factor for carbon metabolism. The TPP riboswitch consensus sequence is highly con-
served and like the coenzyme itself, appears in all three domains of life. The riboswitch controls
both transcription and translation. In E. coli, regulation of thiamine biosynthesis happens at the
49 of 56
translational level: TTP induces an alteration in the 5’ UTR sec-
ondary structure, resulting in sequestration of the Shine-Dalgar-
no sequence and prevention of ribosomal binding. Furthermore,
in B. subtilis TTP can induce an alteration in the 5’ UTR sec-
ondary structure resulting in the formation of a terminator hairpin
structure which stimulates transcription termination.
Riboswitch structures can be detected by in-line probing:

RNAs are incubated either with TPP or without the metabolite
and sequence reads are compared. The dominant pathway by
which RNA degrades involves an “in-line” nucleophilic attack by
the 2′ oxygen on the adjacent phosphorus centre. Cleavage only
takes place if the attacking 2′ oxygen, the phosphate and the
departing 5′ oxygen of the phosphodiester bond approach a linear configuration (“in-line”). The
speed at which spontaneous cleavage occurs depends on the local structural context in which
each RNA linkage exists. Linkages that reside in highly structured regions of a folded RNA (for ex-
ample, a base-paired helix) typically resist cleavage because the relevant atoms are not held in a
linear configuration.
RNA chemical probing uses chemicals that react with un-

paired bases of RNAs such as CMTC modifying uracil, DMS
modifying adenine and cytosine, or kethoxal which modifies
guanine. Their reactivity depends on local RNA structure e.g.
base-pairing or accessibility, meaning that differences in reac-
tivity can therefore serve as a footprint of structure along the
sequence. However, target positions on the RNA can also be
protected from the reagents by binding proteins, enabling ap-
plication of chemical probing to assay protein-binding. In
chemical probing, structured RNA is reacted with probing
reagents which form a covalent adduct on the RNA at the site
of reaction. When RNA is reverse transcribed into a DNA copy
by a reverse transcriptase, the cDNA generated is truncated at
modified positions because the enzyme is blocked by the
adducts. cDNA sequences of various truncated lengths inform
on the frequency of reaction at every base position, which re-
flects the structure profile along the RNA. cDNA sequences are
usually separated and visualised by gel electrophoresis; the
intensity of bands inform on the frequency of observing a trun-
cation at a certain position. Recent approaches use high-
throughput sequencing to achieve a greater throughput and
sensitivity.
https://en.wikipedia.org/wiki/Nucleic_acid_structure_determination
50 of 56
To determine translational repression, mRNA is incubated with TTP and one of the abovemen-
tioned reagents which modify unpaired bases. Thereafter, the extract is purified and mRNA dena-
tured. Subsequently the mRNA is reverse transcribed using primers, which bind various positions
on the mRNA, and radioactively labelled nucleotides. cDNA synthesis arrests at modified bases
generating various cDNA fragments of different lengths which can be separated by gel elec-
trophoresis and visualised on a Southern Blot. In comparison to a cDNA fragments obtained by
reverse transcription of mRNA which had not been provided TTP, band patterns should differ, re-
sembling structural changes in the riboswitch.
WHAT IS NONSENSE MEDIATED DECAY? HOW DO STOP CODONS IN EUKARYOTIC MRNAS LEAD
TO MRNA DECAY? DRAWINGS.
mRNA-surveillance mechanisms engage specialised

mRNA decay pathways such as nonsense-mediated de-
cay (NMD). Following splicing in the nucleus, the exon
junction complex (EJC), which contains UPF3 (up-
frameshift protein essential to the NMD pathway), is asso-
ciated with the transcript, and the resulting messenger ri-
bonucleoprotein is exported to the cytoplasm. In the cyto-
plasm, a second NMD core protein, UPF2, binds to UPF3.
Ribosomes associate and translate the mRNA, but are
stalled on encountering a premature termination codon
(PTC). This results in binding of the SURF complex (com-
prising SMG1, UPF1 and the peptide-release factors eRF1
and eRF3) to the ribosome. UPF1 also binds UPF2, there-
by linking the EJC to the PTC. Phosphorylation of UPF1 by
SMG1 leads to dissociation of eRF1 and eRF3 and binding
of the SMG7 adaptor protein. In S. cerevisiae EJC/SURF
results in decapping of the 5’ end which initiates 5’→3’
mRNA degradation by the 5’→3’ exoribonuclease XRN1. In
mammals, nonsense-mediated decay can lead to two dis-
tinct pathways: endonucleolytic cleavage or deadenylation
of the 3’ poly-A tail. Endonucleolytic cleavage induces ei-
ther 3’→5’ mRNA degradation by the exosome or 5’→3’
decay by XRN1. Deadenylation of the 3’ poly-A tail results
in decapping of the 5’ end followed by 5’→3’ decay by
XRN1.
Other specialised mRNA decay pathways include non-stop decay and no-go decay. Non-stop
decay is engaged upon translation of an mRNA that lacks a stop codon, which results in ribo-
somes “translating” the poly(A) tail, displacing the poly-A-binding protein (PABP) and stalling at
the 3′ end of the mRNA. One model proposes that an adaptor protein that functions as a molecu-
lar mimic of tRNA, binds to the A site on the stalled ribosome to release the transcript, and then
recruits the exosome for 3’→5’ mRNA decay. Another possible pathway is the displacement of
PABP by the translating ribosome which renders the mRNA susceptible to decapping and 5′→3′
decay by the 5′→3′ exoribonuclease XRN1 (in absence of the adaptor molecule). No-go decay is
initiated upon ribosomes stalling within the open reading frame (ORF) on encountering e.g. a
strong secondary RNA structure. Special proteins subsequently bind the transcript near the
stalled ribosome and initiate an endonucleolytic cleavage event near the stall site. This releases
51 of 56
the ribosome and generates two mRNA fragments, each with a free end exposed for exonucle-
olytic decay by the exosome and the 5’→3’ exoribonuclease XRN1, respectively.
Note: nonsense-mediated decay is not to be confused with nonsense suppression:

A suppressor tRNA typically harbours a mutation in the anticodon that changes codons that it recognises. When the
new anticodon corresponds to a termination codon, an amino acid is inserted and the polypeptide chain is extended
beyond the termination codon. This results in nonsense suppression at a site of nonsense mutation, or in read-through
at a natural termination codon. Each termination codon has nonsense suppressors; each type of nonsense codon is
suppressed by tRNAs with mutated anticodons. Suppressor tRNAs compete with wild-type tRNAs that have the same
anticodon to read the corresponding codon(s). Efficient suppression is deleterious because it results in read-through
past normal termination codons. Missense suppression occurs when the tRNA recognises a different codon from usual
so that one amino acid is substituted for another.
DESCRIBE THE MECHANISM OF TRANS-TRANSLATION IN BACTERIA. WHAT IS ITS PURPOSE?
Translation of mRNA generally results in the correct synthesis of a target protein, however there
are various sources of error. Premature termination of transcription, mRNA damage (leads to
cleavage), frameshifting, read-through of a stop codon and stalling on intact mRNA resulting in
cleavage (without the recruitment of EF-P) all lead to the formation of the non-stop complex. The
trans-translation mechanism is a key component of multiple quality control pathways in bacteria
that ensure proteins are synthesised with high fidelity in spite of abovementioned challenges.
trans-translation is performed by a ribonucleoprotein complex composed of tmRNA, a specialised
RNA with properties of both a tRNA and an mRNA, and the small protein SmpB. tmRNA-SmpB
interacts with translational complexes stalled at the 3' end of an mRNA to release the stalled ribo-
somes and mark the nascent polypeptides and mRNAs for degradation. In addition to quality con-
trol pathways, some genetic regulatory circuits use trans-translation to control gene expression.
Diverse bacteria require trans-translation when they execute large changes in their genetic pro-
grams, including responding to stress, pathogenesis, and differentiation.
https://www.ncbi.nlm.nih.gov/pubmed/18557701
The ribonucleocomplex formed by tmRNA and the SmpB protein is recognised and bound by the
ribosome via its tRNA-like domain. Upon accommodation of tmRNA-SmpB, an alanine residue
functioning as a tag is integrated into the nascent peptide; decoding of the tag reading frame re-
sults in proteolysis of the truncated, tagged polypeptide.
52 of 56
WIE KANN MAN NACHWEISEN, DASS IF3 GEGEN NICHT AUG START CODONS DISKRIMINIERT? 2
NACHWEISMETHODEN / ZEICHNUNGEN!
A reporter gene assay on autoregulation of IF3 allows in vivo determination of codon discrimina-
tion. For this purpose, a plasmid encoding the reporter gene lacZ with the start codon AUG is in-
troduced into cell containing wild type IF3; ß-galactosidase can be synthesised. If cells containing
mutant IF3 are transduced with the same plasmid production of ß-galactosidase is possible as
well. However, if a plasmid containing lacZ and an AUU start codon is introduced into cells pos-
sessing wild type IF3, ß-galactosidase cannot be synthesised. IF3 undergoes a conformational
change when bound to a start codon other than AUG, which results in stronger binding preventing
recruitment of the 50S subunit and subsequent transcription. When the same plasmid is intro-
duced into cells possessing a mutant version of IF3, ß-galactosidase can still be produced but
only with a third of the original translational efficiency. Mutant IF3 is no longer able to undergo the
same conformational change which allows wild type IF3 to discriminate between AUG and other
start codons. Therefore, it does not exhibit increased binding affinity to AUU, rather it initiates
translation of ß-galactosidase.
Toeprinting is an in vitro method whereby mRNAs with a known sequence and a RBS are incu-
bated with radioactively labelled primers and nucleotides. Primer extension on mRNA lacking 30S
ribosomes, IF3 or tRNAs results reverse transcription of the mRNA until its 5’ end, producing the
extension signal. However, if the primer is added to mRNA, IF3, 30S ribosome, fMet-tRNA and
radioactively labelled nucleotides, the complex is formed on the AUG codon. If primers are incu-
bated with mRNA, 30S ribosome, tRNAs and nucleotides, the 30S subunit binds mRNA at loca-
tions containing complementary codon sequences of a particular tRNA. Both reactions result in
shorter cDNA fragments as the complex blocks reverse transcription (toeprint signal). If IF3, tRNA,
mRNA, 30S ribosome, labelled nucleotides and primers are incubated together, cDNA fragments
obtained by reverse transcription will be similar in length, as IF3 discriminates against non-cog-
nate complexes, and therefore the complex will be formed on the AUG start codon. DNA frag-
ments obtained by reverse transcription are separated by gel electrophoresis and visualised on a
Southern Blot.
2 MÖGLICHKEITEN, WIE MRNA DURCH IHRE SEKUNDÄRSTRUKTUR AN DER RIBOSOMEN-

BINDESTELLE INAKTIVIERT SEIN KANN NENNEN + SKIZZEN UND NACHWEISMETHODEN DAFÜR!
53 of 56
tional control is mediated by the mutually exclusive
formation of an anti-anti-terminator stem or an in-
trinsic anti-terminator stem. Translational control is
mediated by the mutually exclusive formation of an
anti-anti-RBS stem and or anti-RBS stem.
mRNA can engage various secondary structures

such as inhibitory stem loop structures which can
mask ribosomal binding sites, and thereby effective-
ly inhibit translation initiation. So-called “breathing”
allows mRNA to obtain a single-stranded conforma-
tion enabling binding by the ribosome and subse-
quent transcription. For instance, phage λ possess-
es two translation initiation sites which encode two
different gene products: a lethal hole-former (holin),
essential to the lytic pathway and an inhibitor of
hole-formation enabling lysogeny. The Shine-Dal-
garno sequence of the hole-former is masked by a
stem loop structure, therefore translation of holin is
dependent on a conformational change induced by
binding of S1 to a poly-U sequence. Binding results in a ss-conformation of mRNA by abolishing
the inhibitory stem loop structures allowing induction of holin.
WIE KANN MAN BEWEISEN, DASS DIE BINDUNGSSTELLE DES RIBOSOMALEN PROTEINS S1 UP-
STREAM DER SHINE-DALGARNO SEQUENZ AUF DER PROKARYONTISCHEN MRNA LIEGT?
Co-immunoprecipitation can determine where and how S1 binds: eP complexes offering phage
mRNA fragments induce formation of the translation initiation complex comprising the 30S ribo-
somal subunit, IF3 and mRNA. The initiation complex and mRNA are cross-linked by UV-radiation
and S1 is immunoprecipitated by S1 antibodies. When S1 is purified, cross-linked mRNA is isolat-
ed as well allowing identification of the specific mRNA sequence bound by RNA sequencing. (An
obsolete method employs proteolysis of precipitated complexes and enzymatic sequencing.)
Toeprinting can also be used to determine S1 binding sites: first the primer is extended without
ribosomes or S1, the extension reaches the 5’ end of the mRNA. In a second reaction the internal-
ly initiation complex without S1 is added, the ribosome cannot bind. The third reaction has in-
creasing amounts of S1 added to the initiation complex, whereby the ribosome is now able to
bind to mRNA. The binding is visualised by truncated cDNA (toeprint signal) synthesised by re-
verse transcription.
WIE KANN MAN NACHWEISEN, DASS EIN PROTEIN AUF A) TRANSKRIPTIONELLER, B) TRANSLA-
TIONELLER EBENE ALS REPRESSOR WIRKT?
A reporter gene assay can be implemented to determine the activity of a certain repressor in
vivo. For this purpose, a plasmid encoding a reporter gene under the control of a strong promoter
is introduced into a cell; the reporter gene can be synthesised. However, if cells, first transduced
with a plasmid carrying the reporter gene under the control of a strong promoter, are then further
transduced with a plasmid coding for a certain repressor, reporter gene expression will be inhibit-
ed. To determine whether or not repression happens at a transcriptional or translational level
quantitative real-time PCR can be implemented. mRNA is isolated from both conditions (once
with the repressor, once without the repressor) and reverse transcribed applying qPCR. If mRNA
54 of 56
concentrations of both samples are the same, repression has to happen on a translational level.
However, if mRNA concentrations differ between the samples, the repressor acts on a transcrip-
tion.
Note: I believe that an in vitro transcription assay would be another possible detection method. Transcription is stimu-
lated by an anti-terminator stem loop structure, while formation of an anti-anti-terminator stem loop inhibits transcrip-
tion. Transcriptional analysis can proof transcriptional control by a riboswitch. For this purpose, DNA template, RNA
polymerase and nucleotides are incubated once with a target metabolite and once without. Templates without the me-
tabolite will render long (anti-terminator) fragments, however if metabolites are provided, anti-anti-terminator stem loops
are induced resulting in short fragments.
WIE KANN MAN BIOCHEMISCH NACHWEISEN, WO AN DER 16S RRNA DER INITIATIONSFAKTOR 3
BINDET? (SKIZZEN!)
IF1 is the smallest of the three bacterial translation initiation factors, binding to the ribosomal A
site. Thereby it not only prevents elongator tRNA binding but also controls the conformational dy-
namics of the 30S subunit. Furthermore, by providing the anchoring point for IF2 and IF3 on the
30S subunit IF1 enhances their activity. IF2 is a GTPase that functions to position the initiator
tRNA within the 30S ribosomal initiation complex and promotes its joining with the 50S ribosomal
subunit to form the 70S ribosome. IF3 reduces the affinity of the fMet-tRNAfMet and discriminates
against non-cognate initiation complexes through conformational changes during complex forma-
tion. Due to the localisation of the C-domain of IF3 at the interface side of the platform of the 30S
subunit the factor sterically blocks subunit joining. After release of IF3 a dynamic conformational
switch is introduced by binding of GTP to IF2 which stimulates the rate of subunit joining.
Cryo-electron microscopy (cryoEM) is used to investigate various complex structures and recent
advances have led to new structural insights into many biologically important ribonucleoprotein
(RNP) assemblies, including the spliceosome, ribosome, telomerase, and CRISPR complexes. For
the increasing number of these maps with regions of high-resolution density (<4.0 Å), it is possible
to manually trace atomic coordinates to obtain full-atom models. However, most high-resolution
maps still contain regions of lower resolution in which manual coordinate tracing is not feasible.
For these regions as well as for the sizeable number of maps determined at lower resolution,
atomic coordinates are often obtained by fitting known structures of smaller subcomponents into
the density. This procedure presents a particular challenge for RNA-protein assemblies, as it is
typically difficult to experimentally determine the coordinates of RNA subcomponents in isolation.
The majority of computational methods focus on protein model building and refinement. These
methods, many of which are based on well-established structure prediction algorithms, are able
to build proteins de novo into both high- and lower-resolution maps, but at best can handle the
presence of predetermined RNA structures. In principle, RNA structure prediction algorithms
could be similarly adapted for modelling RNA coordinates de novo into cryoEM maps of RNPs,
but these methods have not yet been expanded to model RNA-protein complexes.
https://www.biorxiv.org/content/biorxiv/early/2018/05/30/332791.full.pdf
Note: I couldn’t find anything useful on the slides or in my notes. But she mentioned cryoEM during structural determi-
nation of the S1 ribonucleoprotein complex, so I thought this could be a possible solution here as well.
55 of 56
GEMEINSAMKEITEN UND UNTERSCHIEDE ZWISCHEN THERMOSENSOR UND RIBOSWITCH? 2 BSP
DER REGULATION DURCH RIBOSWITCHES.
tural change upon ligand binding by the aptamer, and thereby alters gene expression. Transcrip-
Expression of the cobalamin (coenzyme B12) cob operon in Salmonella typhimurium is controlled
by feedback inhibition: high concentrations of the end-product result in repression of protein syn-
thesis. This regulation is conferred mainly at the translational level, and involves a cobalamin-in-
duced folding of an RNA hairpin (coenzyme B12 riboswitch). Normally, the translational en-
hancer activates translation by long-distance interaction with the stem of the RBS hairpin. How-
ever, excessive coenzyme B12 binds the B12 box which results in blocking of the translational
enhancer as well as in a conformational change that sequesters the ribosomal binding site of the
cob mRNA. Thereby binding of the ribosome and subsequent translation initiation is prevented.
Thiamine pyrophosphate (TTP or cocarboxylase) functions as a coenzyme for decarboxylase en-

zymes such as pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, transketolase; thus it is
a key factor for carbon metabolism. The TPP riboswitch consensus sequence is highly con-
served and like the coenzyme itself, appears in all three domains of life. The riboswitch controls
both transcription and translation. In E. coli, regulation of thiamine biosynthesis happens at the
translational level: TTP induces an alteration in the 5’ UTR secondary structure, resulting in se-
questration of the Shine-Dalgarno sequence and prevention of ribosomal binding. Furthermore, in
B. subtilis, TTP can induce an alteration in the 5’ UTR secondary structure resulting in the forma-
tion of a terminator hairpin structure which stimulates transcription termination.
Thermosensors (or RNA thermometers, RNAT) are structural elements located within the 5’ UTR
of protein-coding genes and control its translation by operating as reversible molecular “zippers”
or “switches” that unmask the RBS in response to temperature changes. For instance, E. coli ex-
hibits both forms of conformational change: a heat shock induces increased translation of sigma
32 through “unzipping” of structures which previously masked the start codon. A cold shock re-
sults in a conformational “switch”, refolding the mRNA in such a way that the Shine-Dalgarno se-
quence becomes more accessible and the cold shock protein A (CspA) can be translated.
⋯ Viel Erfolg bei der Prüfung! ⋯
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Genexpression Altfragenausarbeitung

Decker & Moll – WiSe 2018 / 2019

Decker: Multiple Choice

SIGMA-FAKTOREN SIND TEIL DER RNA POLYMERASE IN DER ELONGATIONSPHASE.

OPERONS FÜHREN ZUR ERZEUGUNG POLYCISTRONISCHER RNA.

Transcriptional control in bacteria is mediated by the operon, an array of genes characteristic of

2-KOMPONENTENSYSTEME ÜBERTRAGEN PHOSPHATGRUPPEN.

EUKARYOTISCHE INITIATIONSKOMPLEXE FÜR ALLE RNA POLYMERASEN ENTHALTEN TBP.

ALLE EUKARYOTEN RNA POLYMERASEN WERDEN IN DER C-TERMINALEN DOMÄNE PHOSPHO-

In contrast, initiation of transcription by RNA polymerase I is mediated by the binding of upstream

ELONGATION DER TRANSKRIPTION ERFORDERT VOLLSTÄNDIGE ENTFERNUNG DER NUKLEO-

SOWOHL PROKARYOTEN ALS AUCH EUKARYOTEN VERFÜGEN ÜBER TRANSKRIPTIONSFAK-

TRANSKRIPTIONSFAKTOREN IN DER LEBER REAGIEREN AUF DIE GEGENWART VON LAKTOSE.

Lactase is encoded by a single genetic locus (LCT) on chromosome 2. It is expressed exclusively

TF BINDEN AN DIE RNA POLYMERASE.

MONOMERE TRANSKRIPTIONSFAKTOREN BINDEN PALINDROMISCHE DNA.

ALLE BAKTERIELLEN TF ÜBEN NEGATIVE TRANSKRIPTIONSKONTROLLE AUS.

LACTOSE IST EIN KOAKTIVATOR FÜR DEN LAC-REPRESSOR.

DER GENERELLE TRANSKRIPTIONSFAKTOR TBP ÄNDERT DIE DNA STRUKTUR.

EUKARYOTISCHE TF KÖNNEN DIE ELONGATION DER TRANSKRIPTION REGULIEREN.

ALLE EUKARYOTISCHEN PROMOTOREN HABEN EINE TATA-BOX.

DIE REIHENFOLGE IN DER DIE DER GENERELLEN TRANSKRIPTIONSFAKTOREN AN DEN PRO-

DER GENERELLE TRANSKRIPTIONSFAKTOR TBP IST AN DER INITIATION ALLER EUKARYOTISCH-

CHROMATIN REMODELLING KOMPLEXE ENTHALTEN ATPASEN.

CHIP BZW. CHIP-SEQ. WERDEN ZUM ERSTELLEN VON DURCH HISTON-MODIFIKATIONEN

CHROMATIN REMODELLING KOMPLEXE KÖNNEN HISTONE IN EINEM NUKLEOSOM AUS-

AKTIVE GENE SIND AM AUSTAUSCH VON HISTONEN GEGEN HISTONVARIANTEN ERKENNBAR.

AKTIVE PROMOTOREN SIND AN IHREM HISTON-METHYLIERUNGSMUSTER ERKENNBAR.

Acetylation of histones at lysine residues leads to transcriptional activation, whereas phosphory-

TRANSKRIBTIONSSTART IST GENERELL FREI VON HISTONEN.

ELONGATION DER TRANSKRIPTION BEGINNT NACHDEM HISTONE PHOSPHORYLIERT WURDEN.

Acetylation of histones at lysine residues leads to transcriptional activation, whereas phosphory-

CHROMATINIMMUNPRÄZIPITATION / SEQUENZIERUNG BESTIMMT DIE POSITION VON NUKLEO-

CHROMATIN HUBS KÖNNEN TRANSKRIBIERTE UND NICHTTRANSKRIBIERTE BEREICHE DES

PROTEINE DER TRITHORAX-GRUPPE INDUZIEREN HETEROCHROMATIN.

Epigenetic control of cellular (transcriptional) memory in Drosophila melanogaster: polycomb

HISTON DEACETYLIERUNG BEWIRKT DIE METHYLIERUNG DER DNA.

Decker, Part 2, p. 109

UNTERSCHIEDLICHE SIGMA FAKTOREN DIRIGIEREN RNA POLYMERASE ZU SPEZIFISCHEN GEN-

DNA SCHLAUFEN KÖNNEN ZUR GENEXPRESSION IN BAKTERIEN BEITRAGEN.

MANCHE TRANSKRIPTIONSFAKTOREN BILDEN HOMODIMERE DURCH INTERAKTION VON ZN-

DIE CTD DER RNA POLYMERASE II BINDET TRANSKRIPTIONSAKTIVATOREN.

BEI NIEDRIGEM SAUERSTOFFPARTIALDRUCK LIEGT TF YAP1 REDUZIERT UND INAKTIV VOR.

TF HIF WIRD DURCH NIEDRIGEN SAUERSTOFFPARTIALDRUCK AKTIVIERT.

ALLE STEROIDHORMONREZEPTOREN BINDEN / ERKENNEN DIE SELBE DNA SEQUENZ.

GLUCOCORTICOIDE ERHÖHEN DIE MENGE AN IΚB.

DNA-BINDING REGIONS OF SOME TRANSCRIPTION FACTORS ARE FORMED BY AMPHIPATHIC

PHOSPHORYLIERUNG DES KOAKTIVATOR PGC1ALPHA INHIBIERT DIE AKTIVIERUNG DES TF

HOHE INTRAZELLULÄRE CHOLESTEROLMENGEN FÜHRT ZUR AKTIVIERUNG DES TF SREB.

TRANSCRIPTION STARTS OF TRANSCRIBED GENES ARE FREE OF NUCLEOSOMES.

TRANSCRIPTION FACTORS ARE ABLE TO CAUSE BINDING OF CHROMATIN REMODELLERS TO

A TRANSCRIPTION BUBBLE CONTAINS MRNA-DNA HYBRIDS.

MRNA IS COMPLEMENTARY TO THE CODING STRAND OF DNA.

THE CTCF PROTEIN IS INVOLVED IN THE ISOLATION OF CHROMATIN REGIONS.

A two-component regulatory system serves as a basic stimulus-response coupling mechanism to

"BACKTRACKING" DER RNA POLYMERASE IST WICHTIG FÜR REVERSE TRANSKRIPTION.

DER MEDIATORKOMPLEX BESTEHT AUS BASALEN (GENERELLEN) TRANSKRIPTIONSFAKTOREN.

HISTONCHAPERONE HALTEN NUKLEOSOMEN STABIL, DAMIT SIE VON DER ELONGIERENDEN

Decker: Offene Fragen

WIE NENNT MAN DIE ABGEBILDETE STRUKTUR?

a. Was bedeutet in diesem Zusammenhang LCR und “insulator”?

GENE KÖNNEN DURCH EPIGENETISCHE MECHANISMEN REGULIERT WERDEN.

A nucleosome is a chromatin structure formed by 146bp DNA

WAS WIRD HIER GEZEIGT?