Chapter 7 RNA synthesis and processing

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7RNA Synthesis and

Processing

7 Introduction

• Regulation of gene expression allows cellsto adapt to changes in their environments.

• Regulation of gene expression isresponsible for the distinct activities of themultiple differentiated cell types that makeup complex plants and animals.

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7 RNA Synthesis and Processing

I. Transcription in ProkaryotesA. RNA Polymerase and TranscriptionB. Repressors and Negative Control of

TranscriptionC. Positive Control of Transcription

II. Eukaryotic RNA Polymerases and GeneralTranscription FactorsA. Prokaryotic RNA PolymerasesB. General Transcription Factors and Initiation of

Transcription by RNA Polymerase IIC. Transcription by RNA Polymerases I and III

III. Regulation of Transcription in EukaryotesIV. RNA Processing and Turnover

7 Transcription in Prokaryotes

• An understanding of transcription in E. colihas provided the foundation for studies ofthe far more complex mechanisms thatregulate gene expression in eukaryoticcells.

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7 RNA Polymerase and Transcription

• RNA polymerase is the principal enzymeresponsible for RNA synthesis.

• Like DNA polymerase, RNA polymerase isa complex enzyme made up of multiplepolypeptide chains.

7.1 E. coli RNA polymeraseCore polymerase: 2α, β, & β’ (-σ, sigma)


• A promoter is the DNA sequence to whichRNA polymerase binds to initiatetranscription of a gene.

7.2 Sequences of E. Coli promoters

initiation

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• During elongation, the polymerase remainsassociated with its template while itcontinues synthesis of mRNA.

Figure 7.4

• RNA synthesis continues until thepolymerase encounters a terminationsignal, at which point transcription stops.

Figure 7.6

• After transcription stops, the RNA isreleased from the polymerase, and theenzyme dissociates from its DNA template.

7.4 Transcription by E. coli RNA polymerase

initiation

15 bases

Core polymeraseElongation

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7.6 Transcription termination

≥ 4 UStem-and-loop

ρ (pho) independentρ dependent

Transcription in Prokaryotes

During elongation, the polymerase remainsassociated with its template while itcontinues synthesis of mRNA.

Figure 7.4

RNA synthesis continues until thepolymerase encounters a terminationsignal, at which point transcription stops.

Figure 7.6

After transcription stops, the RNA isreleased from the polymerase, and theenzyme dissociates from its DNAtemplate.

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7 Repressors and Negative Control ofTranscription

• Transcription can beregulated at thestages of bothinitiation andelongation, but mosttranscriptionalregulation inbacteria operates atthe level ofinitiation. 7.7 Metabolism of lactose


• The model of gene regulation wasdeveloped on the basis of experimentsinvolving negative control of the lac operon.

• An operon is a group of adjacent genestranscribed as a single mRNA.

• An operator is a regulatory sequence ofDNA that controls transcription of anoperon.

• A repressor is a regulatory molecule thatblocks transcription.

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7.9 Negative control of the lac operonβ-galactosidase (z)Lactose permease (y)Transacetylase (a)

o: operator


• The central principle of gene regulationexemplified by the lactose operon is that controlof transcription is mediated by the interaction ofregulatory proteins with specific DNAsequences.

cis-acting control elements are regulatorysequences that affect the expression of onlylinked genes on the same DNA molecule.

trans-acting factors are transcriptionalregulatory proteins that affect the expressionof genes located on other chromosomeswithin the cell.

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7 Positive Control of Transcription

• The best-studied example of positive controlin E. coli is the effect of glucose on theexpression of genes that encode enzymesinvolved in the breakdown of other sugars.

• Glucose represses the lac operon even in thepresence of the normal inducer, lactose.

– Figure 7.10– 當G存在時，即使有L，此operon還是關閉的

– 當沒有G時，還要有L，此operon才打開

7.10 Positive control of the lac operon by glucose

• Glucose repressesthe lac operoneven in thepresence of thenormal inducer,lactose.

• Low glucose• cAMP• CAP: catabolite

activator protein -60

α

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7 Eukaryotic RNA Polymerases and GeneralTranscription Factors

• All genes are transcribed by a single coreRNA polymerase in bacteria, buteukaryotic cells contain multiple differentRNA polymerases that transcribe distinctclasses of genes.

• Transcription in eukaryotes takes place onchromatin rather than on free DNA.

• Regulation of chromatin structure is animportant factor in the transcriptionalactivity of eukaryotic genes.

7 Eukaryotic RNA Polymerases

• Eukaryotic cellscontain threedistinct nuclearRNApolymerasesthat transcribedifferent classesof genes.

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7 Eukaryotic RNA Polymerases

• Although all three ofthe nuclear RNApolymerases recognizedifferent promotersand transcribe distinctclasses of genes, theyshare several featuresin common with eachother as well as withbacterial RNApolymerase.

7.11 Structure of yeastRNA polymerase II

12-17 different subunitsNine conserved subunits

7 General Transcription Factors and Initiation ofTranscription by RNA Polymerase II

• mRNA

• Transcription factors are specific proteins thatare required for RNA polymerase II to initiatetranscription.

• General transcription factors are involved intranscription from all polymerase II promotersand therefore constitute part of the basictranscription machinery.

Figure 7.12

CTD: C-terminal domain

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7.12 Formation of a polymerase II transcription initiation complex

7 General Transcription Factors and Initiationof Transcription by RNA Polymerase II

• The TATA box is a regulatory DNAsequence found in the promoters of manyeukaryotic genes transcribed by RNApolymerase II.

• The TATA-binding protein, or TBP, is abasal transcription factor that binds directlyto the TATA box.

• TBP-associated factors, or TAFs, arepolypeptides associated with TBP in thegeneral transcription factor, TFIID.

TAFs + TBP = TFIID

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• Following recruitment of RNA polymeraseII to the promoter, the binding of twoadditional factors—TFIIE andTFIIH—completes formation of theinitiation complex.

7.13 Model of the polymeraseII transcription initiationcomplex


• The Mediator is a large protein complexthat stimulates basal transcription; it alsoplays a key role in linking the generaltranscription factors to the gene-specifictranscription factors that regulate geneexpression.

Figure 7.14

Promoter: General transcription factorsEnhancers: Specific transcription factors

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7.14 RNA polymerase II/Mediator complexes

CTD: polymerase II C-terminal domainTyr-Ser-Pro-Thr-Ser-Pro-Ser27 repeats in yeast & 52 in humans

7 Transcription by RNA Polymerases I and III

• RNA polymerase I is devoted solely to thetranscription of ribosomal RNA genes,which are present in tandem repeats.

7.15 The ribosomal RNA gene

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• The promoters of ribosomal RNA genesspan about 150 base pairs just upstreamof the transcription initiation site.

7.16 Initiation of rDNA transcriptionUBF: Upstream binding factor; SL1: selectivity factor 1

-TATA box


• The genes fortRNAs, 5S rRNA,and some of thesmall RNAsinvolved insplicing andprotein transportare transcribed bypolymerase III.

5S & tRNA promoter is downstream

7.17 Transcription of RNA polymerase III genes

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7 RNA Synthesis and Processing

I. Transcription in Prokaryotes

II. Eukaryotic RNA Polymerases and General TranscriptionFactors

III. Regulation of Transcription in Eukaryotes

cis-acting regulatory sequences: promoters &enhancers

IV. RNA Processing and TurnoverA. Processing of Ribosomal and Transfer RNAsB. Processing of mRNA in EukaryotesC. Splicing MechanismsD. Alternative SplicingE. RNA EditingF. RNA Degradation

7 Regulation of Transcription in Eukaryotes

• An important difference betweentranscriptional regulation in prokaryotesand eukaryotes results from the packagingof eukaryotic DNA into chromatin, whichlimits its availability as a template fortranscription.

• Noncoding RNAs, as well as proteins,regulate transcription in eukaryotic cells viamodifications in chromatin structure.

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7 cis-Acting Regulatory Sequences:Promoters and Enhancers

• Certain cis-acting sequences regulate theexpression of eukaryotic genes.

• Genes transcribed by RNA polymerase IIhave core promoter elements, includingthe TATA box and the Inr sequence, thatserve as specific binding sites for generaltranscription factors.

7.19 A eukaryotic promoter:Thymidine kinase gene of HSV


• Enhancers are transcriptional regulatorysequences that can be located at a sitedistant from the promoter. (Figure 7.20)

• The activity of enhancers depends onneither their distance nor their orientationwith respect to the transcription initiationsite. (Figure 7.21)

7.20 The SV40 enhancer

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7 7.21 Action of enhancers

7 7.21 Action of enhancers (Part 2)

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• DNA looping allows a transcription factorbound to a distant enhancer to interactwith proteins associated with the RNApolymerase/Mediator complex at thepromoter.

7.22 DNA looping

7 RNA Processing and Turnover

• Transcription is usually only the beginningof the series of events required to producea functional RNA.

• Primary transcripts of eukaryotic mRNAsundergo extensive modifications beforethey are transported from the nucleus tothe cytoplasm to serve as templates forprotein synthesis.

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7 Processing of Ribosomal and TransferRNAs

• pre-rRNA is the primary transcript (45S)that is cleaved to form individual ribosomalRNAs—the 28S, 18S, and 5.8S rRNAs ofhigher eukaryotic cells.

7.42 Processing of ribosomal RNAs

Then addition of methylation groups

7 Processing of Ribosomal and TransferRNAs

• pre-tRNA is the primary transcript that iscleaved to form transfer RNAs. (Figure 7.43)

• RNase P is an enzyme capable of catalyzingpre-tRNA cleavage.

• A ribozyme is an enzyme in which RNA ratherthan protein is responsible for catalytic activity.

20 tRNAs

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7.43 Processing of transfer RNAs (Part 1)

7.43 Processing of transfer RNAs (Part 2)

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7 Processing of mRNA in Eukaryotes

• Pre-mRNA is the primary transcript that isprocessed to form messenger RNA in eukaryoticcells. (Figure 7.44)

A 7-methylguanosine cap is what is addedduring the modification of the 5′ end of atranscript.

A poly-A tail is a tract of about 200 adeninenucleotides added to the 3′ ends of eukaryoticmRNAs.

Polyadenylation is the process of adding a poly-A tail to a pre-mRNA.

Figure 7.45

7.44 Processing of eukaryotic messenger RNAs

Special: 異於正常之磷酸雙酯鍵，不易被分解

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7.45 Formation of the 3’ ends of eukaryotic mRNAs

Function of 7-methylguanosine cap & poly-A tail:保護及穩定

7 Splicing Mechanisms

• Pre-mRNA splicing proceeds in two steps.

7.47 Splicing of pre-mRNA Lariat-like intron

5’ splice site

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• Spliceosomes are large complexescomposed of proteins and RNAs.

• Small nuclear RNAs, or snRNAs, arenuclear RNAs that range in size from 50 to200 bases.

• Small nuclear ribonucleoprotein particles,or snRNPs, are complexes of snRNAs withproteins that play central roles in thesplicing process.


• The first step in spliceosome assembly isthe binding of U1 snRNP to the 5′ splice ofpre-mRNA.

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7.48 Assembly of the spliceosome


• Self-splicing is the ability of some RNAs tocatalyze the removal of their own introns.

• Self-splicing RNAs are divided into twoclasses on the basis of their reactionmechanisms.

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7.50 Self-splicing introns

Group I intron Group II intron


• The SR splicing factors bind to specificsequences within exons and act to recruitU1 snRNPs to the 5′ splice site.

7.51 Role of splicing factors in spliceosome assembly

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7 Alternative Splicing

• Alternative splicing is the generation ofdifferent mRNAs by varying the pattern ofpre-mRNA splicing.

• In the sex determination of Drosophila,alternative splicing of the same pre-mRNAdetermines whether a fly is male or female.

7.52 Alternative splicing in Drosophila sex determination

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7 RNA Editing

• RNA editing is an RNA processing eventthat alters the protein-coding sequences ofsome mRNAs.

7.54 Editing of apolipoprotein B mRNA

4536 aa 2152 aa

7 期中考

•期中考 (9th wk)：11/11 (三) 10:20-11:30Chap 1~770 英文選擇1.5 points each

Chapter 7 RNA synthesis and processing

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