Gene Expression Overview

GENE EXPRESSION OVERVIEW

SALWA HASSAN TEAMA 2017


Source:http://medicine.unimelb.edu.au/ehac?a=657716


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.


PROTEIN SYNTHESIS

Protein synthesis is the process in which cells build protein from 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.SALWA HASSAN TEAMA 2017


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.


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

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. SALWA HASSAN TEAMA 2017

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


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.

Polymerase DNA Interaction


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.

TRANSCRIPTION INITIATION

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……………………..

TRANSCRIPTION ELONGATION

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 hybridA 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

TRANSCRIPTION TERMINATION


Source: http://www.personal.psu.edu/rch8/workmg/TxnRNAPolCh10.htm

EUKARYOTICGENE EXPRESSION




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.

The general transcription factors combine with RNA polymerase form a preinitiation complex that is competent to initiate transcription as soon as nucleotide are available.

PREINITIATION COMPLEX


http://en.wikipedia.org/wiki/Wikipedia:Citation_needed

http://en.wikipedia.org/wiki/Wikipedia:Citation_needed

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).


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.

INITIATION

Transcription factors bind to class promoters: e.g.

FACTORS FOR RNA POLYMERASE II (HUMAN CELLS)

Factor

No. ofsubunits

Molecularmass (kDa)

Functions

Functions toRecruit:

TFIID: TBP 1 38 Recognize core promoter (TATA)

TFIIB

TFIID: TAFs 12 15-250 Recognize core promoter (non-TATA); Positive and negative regulation

RNA Pol II?

TFIIA 2 12, 19, 35 Stabilize TBP-DNA binding; Anti-repression

TFIIB 1 35 Select start site for RNA Pol II RNA PolII-TFIIFRNA Pol II 12 10-220 Catalyze RNA synthesis TFIIETFIIF 2 30, 74 Target RNA PolII to promoter;

destabilize non-specific interactions between PolII and DNA

TFIIE 2 34, 57 Modulate TFIIH helicase, ATPase and kinase activities; Directly enhance promoter melting?

TFIIH

TFIIH 9 35-89 Helicase to melt promoter; CTD kinase; promoter clearance?

Source: Roeder, R.G. (1996) TIBS 21: 327-335


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.


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 provided by Example :Comprehensive Yeast Genome Database.

Source: https://labs.fhcrc.org/hahn/Research/polii_res.html


http://mips.gsf.de/proj/yeast/CYGD/db/index.html

http://mips.gsf.de/proj/yeast/CYGD/db/index.html

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.


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.


Source: http://scienceblogs.com/pharyngula/2007/01/16/basics-what-is-a-gene/

PROMOTER CLEARANCEDNA 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.

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.

ELONGATION

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 transcript cleavage occurs cotranscriptionally and presumably preceded cleavage at the polyadenylation site

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.

TRANSLATION

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Translation

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 sourceSALWA HASSAN TEAMA 2017

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).


Large Ribosomal Subunit

Three SitesA 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.


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.)SALWA HASSAN TEAMA 2017

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-tRNAfMet 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.


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.


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

EUKARYOTIC INITIATION


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

Elongation

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.

TERMINATION


http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindtransciption.html

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/

Glossary

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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Gene Expression Overview