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DNA & RNA

METABOLISM
REDARIO C. LAYGO, M.D.
DEPARTMENT OF
BIOCHEMISTRY, MOLECULAR
BIOLOGY
CENTRAL DOGMA IN
MOLECULAR GENETICS
REPLICATION
l USES DNA POLYMERASE
l THREE ALTERNATE SCHEMES OF
REPLICATION :
- CONSERVATIVE
- DISPERSIVE
- SEMI-CONSERVATIVE
CONSERVATIVE
REPLICATION
DISPERSIVE REPLICATION
SEMI-CONSERVATIVE
REPLICATION
REPLICATION ORIGIN
REPLICATION IN E. COLI
STEPS IN REPLICATION
l PRE-PRIMING STAGE
l CHAIN INITIATION
l CHAIN ELONGATION
l REMOVAL OF RNA PRIMER
l DNA LIGASE
Prokaryotic Replication
OriC
l DnaA:
l initiates DNA replication
l a protein that binds to a region of the origin known as the DnaA
box; in E. coli, there are 4 DnaA boxes:
l each of which contains a highly conserved 9 bp consensus
sequence 5' - TTATCCACA - 3'
l binding of DnaA to DnaA boxes causes it to become negatively
supercoiled
l DnaB boxes:
l a region of OriC upstream of the DnaA boxes that become melted
l there are three of these regions:
l each is 13 bp long, and AT-rich
l this region has the consensus sequence 5' - GATCTNTTNTTTT - 3’
l melting of the DnaB boxes requires ATP:
l which is hydrolyzed by DnaA
(245 bp)

Complex of
10-20 DnaA
proteins
CHAIN INITIATION
l USES A SMALL PIECE OF RNA AS
PRIMER FOR DNA SYNTHESIS
l TWO ENZYMES SYNTHESIZE RNA
PRIMER:
- RNA POLYMERASE ON THE LEADING
STRAND
- PRIMASE (PRODUCT OF DNA G GENE)
ON THE LAGGING STRAND
Elongation

l DNA polymerase III holoenzyme is loaded into the DNA and


replication begins
l catalytic mechanism of DNA polymerase III involves the use
of:
l 2 metal ions in the active site:
l the metal ions are general divalent cations that help the 3' OH
initiate a nucleophilic attack onto the a PO4 of the dNTP and
orient and stabilize the negatively charged triphosphate on the
dNTP:
§ nucleophilic attack by the 3' OH on the a PO4 releases PPi, which is then
subsequently hydrolyzed (by inorganic phosphatase) into 2Pi:
§ this hydrolysis drives DNA synthesis to completion
l a region in the active site that discriminate between dNTPs &
NTPs
Elongation

l DNA polymerase III is able to distinguish


between correctly paired bases and incorrectly
paired bases:
l 1st by distinguishing Watson-Crick base pairs

l 2nd, dsDNA (double stranded DNA) in the active site


has a wider major groove and shallower minor groove

l 3rd, the active site makes extensive hydrogen bonds


with the DNA backbone
Elongation

l DNA polymerase III is able to distinguish


between correctly paired bases and incorrectly
paired bases:

l 1st, DNA polymerase III distinguishes Watson-Crick base pairs


through the use of:

l an active site pocket that is complementary in shape to the


structure of correctly paired nucleotides:
§ this pocket has a Tyrosine residue that is able to form van der
Waals interactions with the correctly paired nucleotide
Elongation

l DNA polymerase III is able to distinguish between correctly


paired bases and incorrectly paired bases:

l 2nd, dsDNA (double stranded DNA) in the active site has a


wider major groove and shallower minor groove:
l that permits the formation of hydrogen bonds with the
third nitrogen of purine bases and the second oxygen of
pyrimidine bases.
Elongation

l DNA polymerase III is able to distinguish between correctly


paired bases and incorrectly paired bases:

l 3rd, the active site makes extensive hydrogen bonds with


the DNA backbone
Elongation

l RESULT of the interactions mentioned previously:


l the DNA polymerase III closing around a correctly paired
bases

l if a base is inserted and incorrectly paired, these interactions


could not occur due to disruptions in hydrogen bonding
and van der Waals interactions
Elongation

l DNA template is read in the 3' → 5' direction,


therefore:
l nucleotides are synthesized (or attached to the
template strand) in the 5' → 3' direction
Elongation
l one of the parent strands of DNA is 3' → 5' while the
other is 5' → 3’:
l replication occurs in opposite directions:

l for the parental strand w/ 3’ → 5’ direction:


§ the leading strand, heading toward the replication
fork is synthesized in a continuous fashion, only
requiring one primer

l for the other parental strand w/ 5' → 3’ direction:


§ the lagging strand, heading away from the
replication fork, is synthesized in a series of short
fragments known as Okazaki fragments,
consequently requiring many primers
Elongation
l RNA primers of Okazaki fragments:
l are degraded by RNase H and DNA Polymerase I
(exonuclease)
l the gaps (or nicks) are filled with dNTPs by DNA
Polymerase I
l DNA ligase seals the nicks (creating phosphodiester
bonds)
http://sites.fas.harvard.edu/~biotext/animations/replication1.swf
Termination
l Termination of DNA replication in E. coli is completed
through the use of:
l ter (termination) sequences and the Tus protein:
l these sequences allow the two replication forks to pass through
in only one direction, but not the other

Ter - termination sequences


Tus - termination utilization substance
Products after Termination
l initial product of DNA replication in E. coli produces:
l two catenated or linked circular DNA duplexes:
l each comprising one parental strand and one newly synthesized
strand (by nature of semiconservative replication)
l this catenation seen as two interlinked rings which cannot be
separated:
l Topoisomerase IV in E. coli unlinks or decatenates the two
circular DNA duplexes
Regulation

l mechanism for regulation of DNA replication in E. coli:


l 1st : involves the ratio of ATP to ADP

l 2nd : ratio of DnaA to the number of DnaA boxes

l 3rd : the hemimethylation and sequestering of OriC


Regulation thru ratio of ATP to ADP

l ratio of ATP to ADP indicates:


l the cell has reached a specific size

l readiness to divide.

l this "signal" occurs because in a rich medium the cell:


l will grow quickly

l will have a lot of excess DNA


Regulation thru DnaA-ATP complex

l DnaA-ATP complex is able to initiate replication


l in a fast-growing cell:
l there will be more DnaA-ATP than DnaA-ADP

l the ratio of DnaA to the number of DnaA boxes in the cell is


important because:
l the levels of DnaA are strictly regulated

l 4 DnaA-DnaA dimers are needed to initiate replication

l after completion of DNA replication:


l the above number is halved, thus DNA replication cannot
occur until the levels of DnaA protein increases
Regulation thru Sequestration

l DNA is sequestered to a membrane-binding protein called


SeqA:
l this protein binds to hemi-methylated GATC DNA
sequences:
l this four bp sequence occurs 11 times in OriC
l newly synthesized DNA only has its parent strand methylated
l importance of hemi-methylation:
§ OriC becomes inaccessible to DnaA
§ DnaA binds better to fully methylated DNA than hemi-methylated DNA
l DAM methyltransferase methylates the newly synthesized
strand of DNA only if it is not bound to SeqA
Regulation

l the above mechanisms serve to:


l downregulate DNA replication so that it only occurs once per
cell cycle
l prevents over-replication of DNA

End ---> 72
REPLICATION FORK

5’

5’

3’

5’
SUMMARY OF EVENTS AT
THE REPLICATION FORK
l HELICASE – UNWINDS HELIX; ATP AS
ENERGY SOURCE
l SSB – BINDS TO ssDNA TO PREVENT
REANNEALING
l DNA POL III ELONGATES LEADING
STRAND W/ RNA PRIMER SYNTHESIZED
BY RNA POLYMERASE
l PRIMASE SYNTHESIZES RNA PRIMER ON
THE LAGGING STRAND
SUMMARY OF EVENTS AT
THE REPLICATION FORK
l DNA POL III SYNTHESIZES DAUGHTER
STRAND USING RNA PRIMER ON THE
LAGGING STRAND
l DNA POL III STOPS WHEN IT HITS THE
PREVIOUS RNA PRIMER
l DNA POL I – REMOVES RNA PRIMER AND
REPLACES IT WITH DNA ( NICK
TRANSLATION )
l DNA LIGASE – REPAIRS THE NICK
E.COLI DNA POLYMERASES
l 5’ TO 3’ POLYMERIZATION :
DNA POL I (LARGER DOMAIN), II & III a
l 3’ TO 5’ EXONUCLEASE ACTIVITY :
DNA POL I (SMALLER DOMAIN), II & III ε
l 5’ TO 3’ EXONUCLEASE ACTIVITY :
DNA POL I ONLY
l REPAIR FUNCTION :
DNA POL IV & V
E.coli DNA Polymerases
Turnover # Activity Function
600 - 5’ to 3’ exonuclease as an RNAase H - removes primer from
DNA Pol I 5’ end of previously synthesized Okazaki
fragment
- DNA polymerase catalyze addition of dNTPs to 3’ end of
activity more recently made Okazaki fragment
- intrinsic 3’ to 5’ increases accuracy of gap filling
proofreading
exonuclease
DNA Pol II -30 - 3’ TO 5’ exonuclease damage bypass, DNA repair
activity
DNA Pol III 9000 - 3’ TO 5’ exonuclease synthesizes continuous strand and most
- major replication of the discontinuous (retrograde) strand
polymerase
DNA Pol IV damage bypass

DNA Pol V damage bypass

Turnover # - nucleotides polymerized at 370C/min/mol of enzyme


E.coli DNA Polymerase III
Sub Number of Mr of Genes Function of subunits
unit subunits per subunits
holoenzyme
a 2 129,900 polC(dnaE) Polymerization activity
Core
e 2 27,500 dnaQ(mutD) 3’ to 5’ Proofreading
Polymerase
exonuclease
q 2 8,600 holE Stabilization of e subunit

t 2 71,100 dnaX Stable template binding; Clamp-loading


core enzyme dimerization (g) complex that
loads b subunits
g 1 47,500 dnaX* Clamp loader
on lagging
d 1 38,700 holA Clamp loader strand at each
Okazaki
d’ 1 36,900 holB Clamp loader fragment

c 1 16,600 holC Interaction w/ SSB

y 1 15,200 holD Interaction w/ g and c

b 4 40,600 dnaN DNA clamp required for


optimal processively
*The g subunit is encoded by a portion of the gene for the t subunit, such that theamino-terminal 66% of the t subunit has the same amino acid
sequence as the g subunit. The g subunit is generated by a translational frameshifting mechanism tha leads to preemature translational termination.
Eukaryotic Replication
One Replication during a Single Cell
Cycle

l mechanisms have evolved to regulate, limiting


initiation factors: ORC, Cdt1, and Cdc6:
l after origin firing, Orc1, the largest ORC subunit, is
ubiquitinated and degraded
l phosphorylation of human Orc2 leads to the
dissociation of Orc2-6 from chromatin to further prohibit
re-replication
l Cdc6, once phosphorylated at the onset of S phase, is
rapidly exported from the nucleus
One Replication during a Single Cell
Cycle
l mechanisms have evolved to regulate, limiting initiation
factors: ORC, Cdt1, and Cdc6:
l geminin is a specific inhibitor of Cdt1:
l geminin protein levels increase throughout S phase, blocking
Cdt1 from localizing to replication origins
l Cdt1 and Cdc6 undergo rapid proteasome-dependent
degradation
One Replication during a Single Cell
Cycle
l duplicating the genome once and only once per cell cycle is
controlled at the level of licensing:
l multiple and redundant mechanisms prevent origin assembly
to restrict replication licensing once per cell cycle:
l these controls emphasize the importance of preventing over
replication of chromosomal DNA, in order to preserve genomic
integrity
EUKARYOTIC DNA
POLYMERASES
l DNA POL α – W/ PRIMASE ACTIVITY
– LAGGING STRAND REPLICASE
l DNA POL β – DNA REPAIR
l DNA POL γ – MITOCHONDRIAL DNA REPLICATION
l DNA POL δ – 3’ TO 5’ EXONUCLEASE ACTIVITY (PROOF-
READING)
– UNLIMITED PROCESSIVITY WHEN IN
COMPLEX WITH PCNA (EUKARYOTIC LEADING STRAND
REPLICASE)
l DNA POL ε – DNA REPAIR
– 3’ TO 5’ EXONUCLEASE ACTIVITY
– HIGHLY PROCESSIVE EVEN IN THE ABSENCE OF
PCNA
EUKARYOTIC DNA
POLYMERASES INHIBITORS
l APHIDICOLIN – STRONGLY INHIBITS DNA
POL α , δ , ε
l DIDEOXY NTP’S – STRONGLY INHIBITS
DNA POL β , γ
- WEAKLY INHIBITS DNA POL δ , ε
l N-ETHYLMALEIMIDE – STRONGLY
INHIBITS DNA POL α , γ, δ , ε
DIFFERENCES IN DNA REPLICATION IN
PROKARYOTES AND EUKARYOTES

l PROKARYOTES l EUKARYOTES
-DNA POL I, II, III, IV & V - DNA POL α, β, γ,δ, ε
- POLYMERASES ARE ALSO - NOT ALL POLYMERASES
EXONUCLEASES ARE EXONUCLEASES
- ONE ORIGIN OF - SEVERAL ORIGINS OF
REPLICATION REPLICATION
- LONG OKAZAKI - SHORTER OKAZAKI
FRAGMENTS FRAGMENTS
- NO PROTEINS - HISTONES COMPLEXED
COMPLEXED WITH DNA TO DNA
Control of Replication
An observation:
When a cell in G2 of the cell cycle is fused with a cell in S
phase, the DNA of the G2 nucleus does not begin replicating
again even though replication is proceeding normally in the S-
phase nucleus.
Not until mitosis is completed, can freshly-synthesized DNA be
replicated again.

Two control mechanisms have been identified:


one positive and one negative
This redundancy probably reflects the crucial importance of
precise replication to the integrity of the genome.
Licensing: positive control of
replication
To be replicated, each origin of replication
must be bound by:
• an Origin Recognition Complex of proteins (ORC):
• remain on the DNA throughout the process
• licensing factors:
• accessory proteins
• accumulate in the nucleus during G1 of the cell cycle,
which include:
• Cdc-6 and Cdt-1:
• bind to the ORC
• are essential for coating the DNA with
• MCM proteins. Only DNA coated with MCM proteins
(there are 6 of them) can be replicated.
Licensing: positive control of
replication

Once replication begins in S phase:


• Cdc-6 & Cdt-1 leave the ORCs (the latter by ubiquination and
destruction in proteasomes).
• MCM proteins leave in front of the advancing replication fork
Geminin: negative control of
replication
Geminin:
• protein contained in the G2 nuclei
• prevents assembly of MCM proteins on
freshly-synthesized DNA by:
• probably by blocking the actions of Cdt1
• degraded as the cell completes mitosis so that:
• the DNA of the two daughter cells will be able to respond
to licensing factors
• be able to replicate their DNA at the next S phase
INHIBITORS OF DNA
REPLICATION
l ARA – A & ARA – C
- ANTIVIRAL AGENTS
- INHIBIT DNA REPLICATION BY ACTING
AS NUCLEOTIDE ANALOGUES

l ACTINOMYCIN D
- INTERCALATES WITH DNA
INHIBITORS OF DNA
REPLICATION
l NOVOBIOCIN, OXALINIC ACID, NALIDIXIC
ACID AND COUMERMYCIN
- INHIBITORS OF DNA GYRASE
l SYNTHETIC 2’, 3’ DIDEOXY-NTP
ANALOGUES
- LACK BOTH 2’ AND 3’ HYDROXYL
FUNCTIONS
- FURTHER EXTENSION IS IMPOSSIBLE
REGULATION OF
REPLICATION

l TIMING OF REPLICATION INITIATION


- INVOLVES METHYLATION AND
BACTERIAL PLASMA MEMBRANE
INTERACTION
l SLOW HYDROLYSIS OF ATP BY Dna
PROTEIN
- CYCLES THE PROTEIN BETWEEN
ACTIVE AND INACTIVE FORMS
TRANSCRIPTION

l rRNA ,tRNA, and mRNA ARE SYNTHESIZED


UNDER THE DIRECTION OF DNA TEMPLATES
l HIGHLY SELECTIVE DUE TO SIGNALS
EMBEDDED IN THE NUCLEOTIDE SEQUENCE
OF THE DNA
l PRIMARY RNA TRANSCRIPTS ARE INITIALLY
FAITHFUL COPIES OF CODING STRAND OF DNA
– UNDERGO MODIFICATIONS TO BECOME A
FUNCTIONAL MATURE RNA TRANSCRIPT
DNA TEMPLATE AND RNA
TRANSCRIPT
RNA POLYMERASE
l RESPONSIBLE FOR DNA DIRECTED
SYNTHESIS OF RNA
l IN PROKARYOTES – A SINGLE COMPLEX
ENZYME SYNTHESIZING ALL CELLULAR
RNA
l REQUIRES THE FOLLOWING:
- CODING STRAND OF THE dsDNA
- RIBONUCLEOSIDE TRIPHOSPHATES
( ATP, GTP, UTP, CTP )
E. COLI RNA POLYMERASE

l 465 KILODALTONS

l WITH 5 SUBUNITS – 2 α , 1 β, 1β’, 1σ

l CORE ENZYME – ALL THE SUBUNITS


EXCEPT σ SUBUNIT
TRANSCRIPTION UNIT

l EXTENDS FROM PROMOTER REGION


( NUCLEOTIDE SEQUENCE AT THE
BEGINNING OF A STRETCH OF DNA THAT
IS TO BE TRANSCRIBED ) TO THE
TERMINATOR REGION ( END OF DNA
SEQUENCE TO BE TRANSCRIBED )
STAGES OF TRANSCRIPTION
l BINDING OF RNA POLYMERASE TO DNA
TEMPLATE AT SPECIFIC SITES
l INITIATION
- OPEN PROMOTER COMPLEX
l ELONGATION
l TERMINATION
Coding strand

Non-coding strand
PROMOTER REGIONS OF
PROKARYOTIC DNA

5’ 3’
INHIBITORS OF INITIATION OF
TRANSCRIPTION
l RIFAMYCIN SV
- BINDS TO BETA SUBUNIT OF RNA
POLYMERASE PREVENTING INCOMING
NTP FROM BINDING TO INITIATION SITE
l RIFAMPICIN
- ALLOWS 1ST DINUCLEOTIDE TO FORM
BUT BLOCKS MOVEMENT OF
POLYMERASE ALONG THE DNA
ELONGATION PHASE OF
TRANSCRIPTION

l RNA POLYMERASE – BEGINS TO


SYNTHESIZE A TRANSCRIPT OF THE
DNA SEQUENCE ( ONCE PROMOTER HAS
BEEN RECOGNIZED )
l CARRIED OUT BY CORE ENZYME
l SIGMA SUBUNIT DISSOCIATES AFTER
ABOUT 8 BASES HAVE BEEN
POLYMERIZED
l PAUSES ON G-C RICH AREAS
OH

OH

OH

U
OH

OH

OH

HO

Incoming ribonucleoside triphosphate


OH

U
TERMINATION OF
TRANSCRIPTION
l DETERMINED BY SPECIFIC SEQUENCES IN DNA CALLED -
TERMINATION SITES:
l Rho independent termination:
- INVERTED REPEATS, WHICH ARE TYPICALLY G:C
RICH, SO A STABLE STEM-LOOP STRUCTURE CAN
FORM IN THE TRANSCRIPT VIA INTRACHAIN
BASE-PAIRING

- A NONREPEATING SEGMENT THAT PUNCTUATES


THE INVERTED REPEATS

- A RUN OF 6 TO 8 (7) As IN THE DNA TEMPLATE,


CODING FOR Us IN THE TRANSCRIPT

l Rho dependent termination:


l SPECIAL TERMINATION FACTOR –RHO PROTEIN
Inverted repeats
Rho Dependent Termination

• rho is an ATP-dependent
helicase
• it moves along RNA
transcript, finds the "bubble",
unwinds it and releases RNA
chain
DNA POLYMERASE VS. RNA
POLYMERASE
l DNA POLYMERASE l RNA POLYMERASE
- REQUIRES PRIMER - DOES NOT REQUIRE A
- REQUIRES HD ENZYMES PRIMER
- NO KNOWN
ENDONUCLEASE OR
EXONUCLEASE ACTIVITY
- BINDING ON DNA
TEMPLATE RESULTS
IN LOCAL UNWINDING OF
DNA HELIX
EUKARYOTIC RNA
POLYMERASE
l THREE DISTINCT SPECIES:
- RNA POL I:
-IN THE NUCLEOLUS
- TRANSCRIBES rRNA GENES for the precursor of
28S, 18S and 5.8S
- RNA POL II:
- IN NUCLEOPLASM
- RNAP II
- TRANSCRIBES: - mRNA GENE
- snRNA GENE
- RNA POL III:
- IN NUCLEOPLASM
- TRANSCRIBES tRNA & 5S RNA & SMALL RNAs GENES
EUKARYOTIC GENE
PROMOTER SEQUENCE
Regulatory elements Core region Regulatory elements

CAAT box

- 25 bp

- 80 bp

end
Transcription Initiation by RNA Polymerase II
Requires TBP and the GTFs

l Components of a eukaryotic transcription initiation


complex:
l RNA polymerase II
l six general transcription factors (GTFs)
l an 18-subunit complex called Mediator (or Srb/Med)

l The CTD of RNA Polymerase II:


l anchors Mediator to the polymerase

l Mediator:
l allows RNA polymerase II to communicate with transcriptional
activators bound at sites distal from the promoter
Transcription Initiation by RNA Polymerase II
Requires TBP and the GTFs

l There are six GTFs:


l five of which are required for transcription:
l TFIIB, TFIID, TFIIF, TFIIE, and TFIIH
l the sixth, TFIIA:
l stimulates transcription by stabilizing the interaction of
TFIID with the TATA box
Transcription Initiation by RNA Polymerase II
Requires TBP and the GTFs

l TFIID consists of:


l TBP (TATA-binding protein):
l which directly recognizes:
§ the TATA box within the core promoter
§ a set of TBP-associated factors (TAFs or TAFIIs)

l TBP-TAFII complexes serve as:


l a bridge between the promoter and RNA polymerase II
l some are capable of recognizing core promoters lacking
TATA box
Transcription Initiation by RNA Polymerase II
Requires TBP and the GTFs

l TBP binds to the core promoter through:


l contacts made with the minor groove of the DNA:
l distorting and bending the DNA so that DNA sequences upstream
and downstream of the TATA box come into closer proximity

l Once the TBP/TFIID is bound at the core promoter:


l a complex containing RNA polymerase II and the remaining
GTFs convenes at this site:
l a competent transcription Pre Initiation Complex (PIC) is
established:
§ forming the open complex thereby:
§ transcription begins
TATA box
TFIID
(Complete PIC)
RNA Pol II
Transcription through Nucleosomes
FACT
l a special protein dimer
l stands for "facilitates chromatin transcription"
l partially disassembles the nucleosome immediately
ahead (upstream) of a transcribing RNA Polymerase II
by:
l removing two of the eight histones:
l a single dimer of H2A and H2B histones
l sufficiently loosening the DNA wrapped around that
nucleosome so that RNA Polymerase II can transcribe
through it
l reassembles the nucleosome behind the RNA
Polymerase II by returning the missing histones
PROKARYOTIC &
EUKARYOTIC mRNA
l PROKARYOTIC mRNA l EUKARYOTIC mRNA
- POLYCISTRONIC - MONOCISTRONIC
SEPARATED BY - SYNTHESIZED FROM hn
SPACERS RNA
- 5’ END – LEADER - 5’ END – CAP
SEQUENCES OR 5’ STRUCTURE
UNTRANSLATED - 3’ END – POLY A TAIL
REGIONS
- 3’ END – 3’ - LONGER LIFE SPAN
UNTRANSLATED - MORE STABLE
SEQUENCE - REMOVAL OF INTRONS
- SHORT LIFE SPAN OR INTERVENING
- STABLE ONLY FOR FEW SEQUENCES
MINUTES
5’ CAP OF EUKARYOTIC
mRNA
POLY A TAIL OF EUKARYOTIC
mRNA
Found in all tRNAs

Not found in all tRNAs


Other variable sites are
shown in blue as well

l The modified bases are:


l I = inosine
l mI = methylinosine
l T = ribothymidine
l UH2 = dihydrouridine
l m2G = dimethylguanosine
l y = pseudouridine

Figure 13.10 Structure of tRNA


13-48
Termination of Eukaryotic
Transcription
l different for the 3 different eukaryotic RNA polymerases

l rRNA genes:
l transcribed by RNA Polymerase I
l mRNA, & regulatory RNA genes
l transcribed by RNA Polymerse II
l tRNA, 5S rRNA, & structural RNAs genes
l transcribed by RNA Polymerase III
RNA Polymerase I
l rRNA genes:
l transcribed by RNA Polymerase I
l contain a specific sequence of basepairs (11 bp long in humans;
18 bp in mice):
l recognized by TTF-1 (Transcription Termination Factor for RNA
Polymerase I) - a termination protein
§ binds the DNA at its recognition sequence & blocks further
transcription causing:
§ disengagement of RNA Polymerase I from the template DNA
strand
§ release of newly-synthesized RNA
RNA Polymerase II
l mRNA & regulatory RNA genes
l protein-encoding, structural
l transcribed by RNA Polymerse II:
l lack any specific signals or sequences that direct RNA
Polymerase II to terminate at specific locations
l can continue to transcribe RNA anywhere from a few bp to
thousands of bp past the actual end of the gene
RNA Polymerase II
l the transcript is cleaved at an internal site before RNA
Polymerase II finishes transcribing
l releases the upstream portion of the transcript:
l serving as the initial RNA prior to further processing (the pre-
mRNA in the case of protein-encoding genes)
l the cleavage site is considered the "end" of the gene
l remainder of the transcript is digested by a 5'-exonuclease
(called Xrn2 in humans) while it is still being transcribed by the
RNA Polymerase II
RNA Polymerase II
l the 5'-exonulease "catches up" to RNA Polymerase II by
digesting away all the overhanging RNA:
l helps disengage the polymerase from its DNA template strand:
l finally terminating that round of transcription
RNA Polymerase II
l in protein-encoding genes, the cleavage site which
determines the "end" of the emerging pre-mRNA occurs
between:
l an upstream AAUAAA sequence
l a downstream GU-rich sequence separated by about 40-60
nucleotides in the emerging RNA
l once both of these above sequences have been transcribed
l CPSF protein (Cleavage & Polyadenylation Specificity Factor):
§ in humans, binds the AAUAAA sequence
l CstF protein (Cleavage Stimulatory Factor or Cleavage
Stimulation Factor):
§ in humans, binds the GU-rich sequence
CPSF & CstF Proteins
l CPSF & CstF proteins:
l form the base of a complicated protein complex at the regions of
AAUAAA and GU-rich sequences before CPSF cleaves the nascent pre-
mRNA at a site 10-30 nucleotides downstream from the AAUAAA site

l Poly(A) Polymerase enzyme which catalyzes the addition of a 3'


poly-A tail on the pre-mRNA:
l is part of the complex that forms with CPSF and CstF
RNA Polymerase III
l tRNA, 5S rRNA, and structural RNAs genes:
l transcribed by RNA Polymerase III
l have a not-entirely-understood termination signal
l RNAs transcribed by RNA Polymerase III:
l have a short stretch of four to seven U's at their 3' end:
§ this U’s somehow triggers RNA Polymerase III to both:
§ release the nascent RNA
§ disengage from the template DNA strand
INHIBITION OF TRANSCRIPTION
BY ANTIBIOTICS
l TYPE I ANTIBIOTICS :
- BIND TO RNA POLYMERASE – DIRECT
INACTIVATION OF ENZYME
RIFAMYCIN - BINDS TO BETA SUBUNIT OF
PROKARYOTIC RNA POLYMERASE – INHIBIT
CHAIN INITIATION
STREPTOLYDIGIN – BINDS ALSO TO BETA
SUBUNIT & INHIBITS PROGRESSIVE EVENTS
OF ELONGATION AFTER INITIATION OF CHAIN
GROWTH
INHIBITION OF TRANSCRIPTION
BY ANTIBIOTICS
l TYPE II ANTIBIOTICS:
- INDIRECT INACTIVATION OF RNA
POLYMERASES BY BINDING TO DNA
TEMPLATE
ACTINOMYCIN D – NONCOVALENTLY
BINDS TO DUPLEX DNA BY
INTERCALATION – INTERFERES W/
MOVEMENT OF RNA POLYMERASE
ALONG DNA
INHIBITION OF TRANSCRIPTION
BY ANTIBIOTICS

l TYPE III ANTIBIOTICS:


- INDIRECT INHIBITORS – ACT IN
PROKARYOTES BY INHIBITING DNA
GYRASE – COUMERMYCIN
NALIDIXIC ACID
NOVOBIOCIN
INHIBITION OF TRANSCRIPTION
BY ANTIBIOTICS
l CORDYCEPIN - 3’ DEOXYADENOSINE
- ADENOSINE ANALOGUE – LACKS 3’
HYDROXYL GROUP
- INHIBITS BACTERIAL RNA SYNTHESIS
- ITS ADDITION TO 3’ END OF RNA –
PREVENTS RNA CHAIN’S FURTHER
ELONGATION