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GENE EXPRESSION OVERVIEW

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GENE EXPRESSION

Gene expression process by which a genes information is


converted into the structures and functions of a cell by a process
of producing a biologically functional molecule of either protein
or RNA (gene product) is made.

Gene expression is assumed to be controlled at various points in


the sequence leading to protein synthesis.

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PROTEIN SYNTHESIS

process in which cells build protein from


Protein synthesis is the
information in DNA in two major steps:
Transcription
Synthesis of an RNA that is complementary to one of the
strands of DNA according to instruction stored along a specific
sequence (a gene) of a DNA molecule.
Translation
Ribosomes read a messenger RNA and make protein according
to its instruction.
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TRANSCRIPTION
Transcription

Transcription is a vital control point in the expression of many genes.

RNA polymerase directs transcription. RNA polymerase is the signal


that control transcription.
Transcription proceeds in the 5' 3' direction.

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Transcription Enzymes

Eukaryotic nuclei contain three RNA polymerases. RNA polymerase I


found in the nucleolus; RNA polymerase II &III are located in the
nucleoplasm..
The three nuclear RNA polymerase have different roles in transcription:
Polymerase I makes a large precursor to the major rRNA (28S,18S and
5.8S rRNA in vertebrates).
Polymerase II synthesizes hnRNAs, which are precursors to mRNAs. It
also make most small nuclear RNAs (snRNAs).).
Polymerase III makes the precursor to 5SrRNA, the tRNAs and several
other small cellular and viral RNAs.
Prokaryotes have one type of RNA polymerase for all types of RNA.
PROKARYOTIC
GENE EXPRESSION

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Robert F. Weaver. Molecular Biology. 600 Pages. Fourth Edition. McGraw-Hill International Edition. ISBN 978-
0-07-110216-2.
Prokaryote Genes Are Grouped in Operons

Transcription has three phases: initiation, elongation,


termination. The following is an outline of the three step in
bacteria
The key player in the transcription process is RNA
polymerase. The E- coli enzyme is composed of a core, which
contains the basic transcription machinery, and a - factors
which directs the core to transcribe specific gene.
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TRANSCRIPTION INITIATION
Represented as four steps:
Formation of a closed promoter complex.
Conversion of the closed promoter to an open
promoter complex.
Polymerizing the first few nucleotides while
the polymerase remain at the promoter.
Promoter clearance

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Hinkle and Chamberlin summarized this hypothesis for polymerase DNA
interaction: RNA polymerase holoenzyme binds loosely to DNA at first at the
promoter (closed promoter complex). It either binds initially at a promoter or
scans along the DNA until it find one.
Then polymerase melt a short region of DNA at the promoter to form an open
promoter complex in which polymerase is bound tightly to the DNA.
Conversion from loosely bound polymerase (closed promoter complex) to tightly
bound polymerase (open promoter complex) requires - factor
The - factor allows initiation of transcription by causing the RNA polymerase
holoenzyme to bind tightly to a promoter.

Robert F. Weaver. Molecular Biology. 600 Pages. Fourth Edition. McGraw-Hill International Edition. ISBN 978-
0-07-110216-2.
TRANSCRIPTION INITIATION

On binding to a promoter, RNA polymerase causes the unwinding or localized


melting of the DNA double helix which expose at least 12 bases on the
template (estimate 10-17).
This is followed by initiation of RNA synthesis at this starting point.
The transcription bubble moves with the polymerase, exposing the template
strand so it can be transcribed.
The RNA polymerase starts building the RNA chain; it assembles
ribonucleotides triphosphates: ATP; GTP; CTP and UTP into a strand of
RNA. The first, or initiating substrate is usually a purine nucleotide.
After the first nucleotide is in place, the polymerase joins a second nucleotide
to the first, forming the initial phosphodiester bond in the RNA chain.
After has participated in initiation, it appears to dissociate from the core
polymerase. can be reused by different core polymerase.
RNA polymerase directs the sequential binding of
riboncleotides to the growing RNA chain in the 5' - 3'
direction.
RNA polymerase moves along DNA template, and the
bubble of melted DNA moves with it and This melted
region exposes the bases of DNA one by one so they can
pair with the bases of incoming ribonucleotide.
Each ribonucleotide is inserted into the growing RNA
strand following the rules of base pairing. As soon as the
transcription machinery passes, the two DNA strands wind
around each other again. This process is repeated till the
desired RNA length is synthesized..
Some regions on the DNA that signal termination
(terminators) are recognized by RNA polymerase and work
in conjunction with it to loosen the association between
RNA product and DNA template. The RNA dissociate from
RNA polymerase and DNA stop transcription.
Two kinds of terminator:
Intrinsic terminators, function with the RNA polymerase

by itself without help of other proteins.


Rho dependent termination
Intrinsic terminators have two important elements:
An inverted repeat that allows a hairpin to form at the end of the transcript to
destabilize the RNA-DNA hybrid
A string of T ' s in the nontemplate strand that results in a string of weak rU-dA base
pairs holding transcript to template. Together, these elements cause the polymerase
to pause and the transcript to be released.

Rho- dependent terminators consist of an inverted repeat, which can cause a hairpin
to form in the transcript, but no string of T's. Rho binds to growing transcript, follow
the RNA polymerase, catches the polymerase when it pauses at the hairpin, and
release the transcript from the DNA polymerase complex by unwinding the RNA-
DNA hybrid. This terminate transcription

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EUKARYOTIC
GENE EXPRESSION

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Robert F. Weaver. Molecular Biology. 600 Pages. Fourth Edition. McGraw-Hill International Edition. ISBN 978-
0-07-110216-2.
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The general transcription factors combine with RNA
polymerase form a preinitiation complex that is competent to
initiate transcription as soon as nucleotide are available.

First, an RNA polymerase along with general transcription


factors binds to the promoter region of the gene to form a
closed complex called the preinitiation complex.

Preinitiation complex contains:


Core Promoter Sequence
Transcription Factors
RNA Polymerase
Activators and Repressors.
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Three types of RNA polymerase have different structure and they
transcribe different classes of genes.

The class II preinitiation complex contains polymerase II and six


transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH. The class
general transcription factors and RNA polymerase bind in specific order to
the growing preintiation complex (at least in vitro).

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Transcription starts upstream from the first coding sequence at the transcription
initiation site.
RNA polymerase recognizes a promoter. Eukaryotic Promoter lies upstream of
the gene. There are several different types of promoter found in human genome,
with different structure and different regulatory properties class/I/II/III.
One important promoter sequence is the TATA box, a conserved region rich in
adenines and thymines, approximately 20-30 bp upstream of the start site of
transcription. The TATA box appears to be important for determining the position
of the start of transcription.
The assembly of the preinitiation complex on each kind of eukaryotic promoter
e.g. (class II promoters recognized by RNA polymerase II) begins with the
binding of an assembly factor to the promoter, this factor is TBP, but other
promoters have their own assembly factors. Even if TBP is not the first bound
assembly factors at a given promoter, it becomes part of the growing preintiation
complex on most known promoters and serves an organizing function in building
the complex.
This tight binding involves the formation of an open promoter complexes in
which the DNA at the transcription start site has melted to allow the polymerase
to read it.
Transcription
factors bind to
class
promoters: e.g.
FACTORS FOR RNA POLYMERASE II (HUMAN CELLS)

No. of Molecular Functions to


Factor subunits mass (kDa) Functions Recruit:
TFIID:TBP 1 38 Recognize core promoter TFIIB
(TATA)
TFIID:TAFs 12 15-250 Recognize core promoter (non- RNA Pol II?
TATA); Positive and negative
regulation
TFIIA 2 12, 19, 35 Stabilize TBP-DNA binding;
Anti-repression
TFIIB 1 35 Select start site for RNA Pol II RNA PolII-TFIIF
RNA Pol II 12 10-220 Catalyze RNA synthesis TFIIE
TFIIF 2 30, 74 Target RNA PolII to promoter;
destabilize non-specific
interactions between PolII and
DNA
TFIIE 2 34, 57 Modulate TFIIH helicase, TFIIH
ATPase and kinase
activities;Directly enhance
promoter melting?
TFIIH 9 35-89 Helicase to melt promoter;
CTD kinase; promoter
clearance?
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The Mediator Complex And The Polymerase II Holoenzyme

Mediators, another collection of proteins and can be considered as a general


transcription factor, because it is a part of most, if not all class II preintiation
complexes.
Mediator is not required for initiation per se, but it is required for activated
transcription.
Mediator was first discovered in yeast, and found to contain about 20
polypeptides.
Human mediator was discovered, it is a very large complex of over 20
polypeptides, few have clear homology to yeast mediator.
RNA polymerase II holoenzyme contains RNA polymerase, a subset of general
transcription factors and the mediator complex.
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Source: https://labs.fhcrc.org/hahn/Research/polii_res.html

Model of RNA Polymerase II Transcription Initiation Machinery.The machinery depicted here encompasses over 85 polypeptides in ten
(sub) complexes: core RNA polymerase II (RNAPII) consists of 12 subunits; TFIIH, 9 subunits; TFIIE, 2 subunits; TFIIF, 3 subunits; TFIIB,
1 subunit, TFIID, 14 subunits; core SRB/mediator, more than 16 subunits; Swi/Snf complex, 11 subunits; Srb10 kinase complex, 4
http://www.bio.davidson.edu/courses/genomics/2002/james/favoriteyeastproteins.htmsubunits; and SAGA, 13 subunits. This figure
.Comprehensive Yeast Genome Database: provided by Example

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Enhancers

The activity of many promoters are greatly increased by


sequence called enhancers which can exert their stimulatory
actions over distances of several thousands base pairs.
Enhancers can be upstream, downstream or even in the midst
of transcribed gene.

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Activators
The general transcription factors by themselves dictate the starting point
and direction of transcription but they are sponsoring a very low level of
transcription (basal level of transcription).
Activators (gene specific transcription factors) can provide
EXTRABOOST in transcription. Activators can bind to enhancers and
also permits cells to control expression of their genes.
Eukaryotic activators recruit RNA polymerase to promoters but not directly
as prokaryotic activators. Eukaryotic activators stimulate binding of
general transcription factor and RNA to the promoter.
Other finding suggested that, the activator recruited the intact holoenzyme
to the promoter rather than building it up step by step on the promoter.
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DNA helicase of TFIIH is causes further unwinding of the DNA expanding the
transcription bubble With energy provided by ATP. This expansion release the
.polymerase and allow it to clear promoter

ELONGATION
Creation of transcription bubbles
Continuous addition of NTPs, the elongation complex continuous
elongating the RNA.
TBP and TFIIB remains at the promoter. TFIIE and TFIIH are not needed
for elongation and dissociate from the elongation complex.
TERMINATION
The region downstream of the polyadenylation site is essential for
termination.
Cleavage of the nascent transcript at multiple sites downstream of the
polyadenylation sites downstream of polyadenylation site is required for
termination.
The product is immature mRNA Pre mRNA
(Primary transcript)

The primary product of RNA transcription; the


hnRNAs contain both intronic and exonic
sequences.

These hnRNAs are processed in the nucleus to


give mature mRNAs that are transported to the
cytoplasm where to participate in protein
synthesis.
RNA PROCESSING
Capping
The cap structure is added to the 5' of the newly transcribed mRNA
precursor in the nucleus prior to processing and subsequent transport of the
mRNA molecule to the cytoplasm. The 5' cap is a 7-methylguanosine
triphosphate.
Splicing
Step by step removal of introns and joining of remaining exons; it takes
place on a special structure called spliceosomes.
Addition of poly A tail Synthesis of the poly (A) tail involves cleavage of
its 3' end and then the addition of about 40- 200 adenine residues to form a
poly (A) tail. Poly A tail appears to increase stability of the resulting
polyadenylated RNA.
RNA PROCESSING
Alternative Splicing

Alternative splicing: is a very common phenomenon in higher


eukaryotes. It is a way to get more than one protein product out
of the same gene and a way to control gene expression in cells.
TRANSLATION

Translation is the process by which ribosomes read the


genetic message in the mRNA and produce a protein
according to message instruction.
Click icon to add picture

TRANSLATION

Translation

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The overall scheme is similar in bacteria and eukaryotes,
but there are significant difference, especially added
complexity of the eukaryotic translation initiation system
Requirement For Translation

Ribosomes
tRNA
mRNA template
Amino Acids
Initiation factors
Elongation factors
Termination factors
Aminoacyl tRNA synthetase
Energy source

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Ribosomes
Factory for protein synthesis.
Composed of ribosomal RNA and ribosomal proteins;
known as a Ribonucleoprotein (RNP).
Translate messenger RNA (mRNA) to build polypeptide
chains using amino acids delivered by transfer RNA
(tRNA).

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Large Ribosomal Subunit

Three Sites
A site bind to an aminoacyl
tRNA (tRNA bound to an amino
acid).
P site bind a peptidyl tRNA (a
tRNA bound to peptide being
synthesized).
E site binds a free tRNA before
it is exist the ribosome.
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Preparatory Steps For Protein Synthesis

First, activated aminoacyl tRNA synthetase join amino acid to


their specific tRNA.
Second, ribosomes must dissociate into subunits at the end of
each round of translation.
The protein synthesis occur in 3 phases:
Accurate and efficient initiation occurs, the ribosomes binds to the
mRNA, and the first amino acid attached to its tRNA.
Chain elongation, the ribosomes adds one amino acid at a time to
the growing polypeptide chain
Accurate and efficient termination, the ribosomes releases the
mRNA and the polypeptide.
Translation Initiation

The initiation phase of protein synthesis requires many Initiation Factors.


The small subunit of the ribosome binds to a site "upstream" of the start of
the message.
The small subunit of the ribosome proceeds downstream (5' - 3') until it
encounters the start codon AUG.
Then the small subunit of the ribosome is joined by the large subunit and a
special initiator tRNA.
The initiator tRNA binds to the P site on the ribosome.
In eukaryotes, initiator tRNA carries methionine (Met). Bacteria use (fMet.)
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Translation (Initiation In Bacteria)

Dissociation of the 70s ribosomes into 50s and 30s subunits under the influence of IF1
Binding of IF3 to the 30S subunit, which prevents reassociation between the ribosomal
subunits.
Binding of IF2,IF2 and GTP alongside IF3.
Binding of mRNA and fMet-tRNAfMet to form the 30S initiation complex. These two
components can apparently bind in either order, but IF2 sponsors fMet-tRNA fMet
binding, and IF3 sponsors mRNA binding. In each case, the other initiation factors also
help.
Binding of the 50S subunit, with loss of IFI and IF3
Dissociation of IF2 from the complex, with simultaneous hydrolysis of GTP. The
product is 70 S complex ready to begin elongation.

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Prokaryotic Initiation

The initiation codon in prokaryotes is usually AUG, but it can also be GUG, or more
rarely, UUG.
The initiating aminoacy1-tRNAfMet. Is N-formy1-methionine (fMet) is therefore the
first amino acid incorporated into a polypeptide, but it is frequently removed from the
protein during maturation.
The 30S initiation complex is formed from a free 30S ribosomal subunit plus mRNA and
fMet-tRNAfMet.
Binding between the 30S prokaryotic ribosomal subunit and the initiation site of an
mRNA depends on base pairing between a short RNA sequence called the Shine-
Dalgarno sequence just upstream of the initiation codon, and a complementary sequence
at the 3- end of the 16S rRNA. This binding is mediated by IF3, with help from IF1 and
IF2. All three initiation factors have bound to the 30S subunit by this time.

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EUKARYOTIC INITIATION

Eukaryotic 40S ribosomal subunits, together with the initiator


tRNA (tRNAiMet), generally locate the appropriate start codon
by binding to 5-cap of an mRNA and scanning downstream
until they find the first AUG in a favorable context.

The best context contains a purine at position -3 and a G at


position +4 where the A of the AUG is +1.

In 5-10% of the cases, the ribosomal subunits will bypass the


first AUG and continue to scan for a more favorable. one

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The elongation processes in bacteria and eukaryotes are very
similar
To begin elongation, another amino acid needed to join the first. The second amino acid
arrives bound to tRNA, which otherwise empty. This step requires a protein elongation
factor known as EF-TU and GTP.
Peptide bond formation: An enzyme peptidyl transferase transfers the fMet from its tRNA
in the P site to the aminoacyl tRNA in the A site. The whole assembly in the A site is
dipeptidyl tRNA, and deacylated tRNA remains in the P site (tRNA without its amino
acids).
Translocation: the mRNA with its peptidyl tRNA attached in the A site moves one codon`s
length to the left lead to; the deacylated tRNAin the P sites leaves the ribosomes via the E
sites, the dipeptidyl tRNA in the A site, along with its corresponding codon moves into the
P site. Translocation requires an elongation factor called EF-G in bacteria ; EF-2 in
eukaryotes plus GTP.
The process repeats itself to add another amino acids, and continuous over and over until
the ribosomes reaches the last codon in the message. When the polypeptide complete , it is
a time for chain termination
Translational termination requires specific protein factors identified as releasing factors,
RFs in E. coli and eRFs in eukaryotes.
The signals for termination are the same in both prokaryotes and eukaryotes. These
signals are termination codons present in the mRNA. There are 3 termination codons,
UAG, UAA and UGA.
Prokaryotic translation termination is mediated by three factors: RF1,RF2 and RF3. RF1
recognizes the termination codon UAA and UAG; RF2 recognizes UAA and UGA. RF3 is
a GTP binding protein that facilitate binding of RF1 and RF2 to the ribosome.
Eukaryotes have two release factors: eRF1 which recognizes all three termination codons,
and eRF3, a ribosome dependent GTPase that helps eRF1 release the finished polypeptide.
After multiple cycles of elongation and polymerization of specific amino acids into protein
molecules, a nonsense codon = termination codon of mRNA appear in the A site. The is
recognized as terminal signal by releasing factors which cause the release of the newly
synthesized protein from the ribosomal complex.

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Reading the instruction
means translating the code
in the RNA from bases
building block of DNA and
RNA to amino acids
Control Of Gene Expression In Eukaryotic
Control of Gene Expression
Antibiotic Inhibit Protein Synthesis
. Alleles are forms of the same gene with small differences in their sequence of DNA bases
Alternative splicing is a very common phenomenon in higher eukaryotes. It is a way to get more than one protein
.product out of the same gene and a way to control gene expression in cells
Exon: a segment of a gene that is represented in the mature RNA product. Individual exons may contain coding
.DNAand/or noncoding DNA (untranslated sequences)
. Introns (intervening sequence) (A noncoding DNA sequence ): Intervening stretches of DNA that separate exons
Primary transcript: The initial production of gene transcription in the nucleus; an RNA containing copies of all exons
.and introns
RNA gene or non-coding RNA gene: RNA molecule that is not translated into a protein. Noncoding RNA genes produce
transcripts that exert their function without ever producing proteins. Non-coding RNA genes include transfer RNA
(tRNA) and ribosomal RNA (rRNA), small RNAs such as snoRNAs, microRNAs, siRNAsand piRNAs and lastly long
.ncRNAs
Enhancers and silencers: are DNA elements that stimulate or depress the transcription of associated genes; they rely on
tissue specific binding proteins for their activities; sometimes a DNA elements can act either as an enhancer or silencer
.depending on what is bound to it
. Activators: Additional gene-specific transcription factors that can bind to enhancer and help in transcription activation
Open reading frame (ORF): A reading frame that is uninterrupted by translation stop codon (reading frame that contains
. a start codon and the subsequent translated region, but no stop codon)
Directionality: in molecular biology, refers to the end-to-end chemical orientation of a single strand of nucleic acid. The
chemical convention of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5' end and a 3' end
( "five prime end" and "three prime end"). The relative positions of structures along a strand of nucleic acid, including
genes, transcription factors, and polymerases are usually noted as being either upstream (towards the 5' end) or
.downstream (towards the 3' end)
.Reverse Transcription: Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA
.Pseudogenes. DNA sequences that closely resemble known genes but are nonfunctional
.Transcription bubble: region containg RNA polymerase, DNA and newly formed RNA
/More:http://www.ncbi.nlm.nih.gov/books/NBK7584
References And Further Reading
Ali Khalifa. Applied molecular biology; eds: ( Fathi Tash and Sanna Eissa). 109 pages. Egypt. University Book Center. 2002. Available in paper copy
from the publisher

Daniel H. Farkas. DNA Simplified: The Hitchhiker's Guide to DNA. 110 pages. Washington, DC: AACC Press, 1996, ISBN 0-915274-84-1. Available
in paper copy from the publisher

Innis, David H. Gelfand, John J. Sninsky. PCR Applications: Protocols for Functional Genomics: 566 pages. Academic Press; 1 edition (May 17,
1999). ISBN:0123721865. Available in paper copy from the publisher

Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of the cell. 1392 pages.Garland
Science; 5 edition (November 16, 2007).ISBN. 9780815341055. Available in paper copy from the publisher

Robert F. Mueller, Ian D. Young. Emery's Elements of Medical Genetics: Publisher:Churchill Livingstone. 1995 ISBN. 044307125X.
Available in paper copy from the publisher.

Robert F. Weaver. Molecular Biology. 600 Pages. Fourth Edition. McGraw-Hill International Edition. ISBN 978-0-07-110216-2.
Available in paper copy from the publisher

William B. Coleman, Gregory J. Tsongalis. Molecular Diagnostics. For the Clinical Laboratorian: 592 pages.Humana Press; 4th Printing. edition
(August 15, 2005). ISBN 1588293564... Available in paper copy from the publisher.

Eukaryotic promoter . Internet. Available from; http://www.patentlens.net/daisy/promoters/242/g1/250/1346.html

Transcription factor. Available from. Fred Hutchinson Cancer Research Center

Transcription factor . Internet. Table. Available from; http://oregonstate.edu/instruction/bb492/lectures/EuTranscriptionI.htm

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Thank you

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