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1、Transcription and Post-transcription Modification,Central dogma,WHICH CAME FIRST,THE chicken or the egg?The biological silences have a variation:which came first,DNA or protein?You see,among the many tasks performed by proteins is assembling DNA molecules.But DNA contains the information needed to m
2、ake proteins.So which came first?,RNA and RNA world,Walter Gilbert1980 Nobel Prize,Origin-of-LifeTheories RNA has the ability to act as both genes and enzymes,The synthesis of RNA molecules using DNA strands as the templates so that the genetic information can be transferred from DNA to RNA.Four sta
3、ges:Initiation,Elongation,Termination,Post-transcriptional modification,Transcription,Only the template strand is used for the transcription,but the coding strand is not.Both strands can be used as the templates.The transcription direction on different strands is opposite.This feature is referred to
4、 as the asymmetric transcription.,Asymmetric transcription,Template,The template strand is the strand from which the RNA is actually transcribed.It is also termed as antisense strand.The coding strand is the strand whose base sequence specifies the amino acid sequence of the encoded protein.Therefor
5、e,it is also called as sense strand.,Both processes use DNA as the template.Phosphodiester bonds are formed in both cases.Both synthesis directions are from 5 to 3.,Similarity between replication and transcription,Differences between replication and transcription,The whole genome of DNA needs to be
6、replicated,but only small portion of genome is transcribed in response to the development requirement,physiological need and environmental changes.DNA regions that can be transcribed into RNA are called structural genes.,What do the most DNA do in deed?,General concepts of Transcription Process,Thre
7、e phases:initiation,elongation,and termination.The prokaryotic RNA-pol can bind to the DNA template directly in the transcription process.The eukaryotic RNA-pol requires co-factors to bind to the DNA template together in the transcription process.,Transcription bubble,Transcription in prokaryotes,Tr
8、anscription Unit,Optimal Promoter,The 35 sequence is used for initial recognition,and the 10 sequence is used for the melting reaction that converts a closed complex to an open complex.,During transcription,the bubble is maintained within bacterial RNA polymerase,which unwinds and rewinds DNA,mainta
9、ins the conditions of the partner and template DNA strands,and synthesizes RNA.,Bacterial RNA Polymerases,A single type of RNA polymerase is responsible for almost all synthesis of mRNA,rRNA and tRNA in a eubacterium.,About 7,000 RNA polymerase molecules are present in an E.coli cell.Probably 2,0005
10、,000 enzymes are synthesizing RNA at any one time,the number depending on the growth conditions.,How does RNA polymerase work?,RNA pol b subunit is the target of Rifamycin,How many sigma factors exist in E.coli?,How does transcription initiate?,Four stages of Transcription,Termination,The terminator
11、 is in the transcript,not the DNAForms a hairpinSelf-complementaryThe hairpin structure is the signal for terminationRho()-dependent vs.-independent,Intrinsic terminators-independent,An inverted repeat that allows a hairpin to form at the end of the transcriptsA string of Ts in the nontemplate stran
12、d that results in a string of weak rU-dA base pairs holding the transcript to the template strand,Rho factor pursues RNA polymerase along the RNA and can cause termination when it catches the enzyme pausing at a rho-dependent terminator.,Termination of-dependent,Transcription in Eukaryotes,RNA polym
13、erases in Eukaryotes,RNA polymerase I transcribes rRNA RNA polymerase II transcribes hnRNA（heterogeneous nuclear RNA）RNA polymerase III transcribes tRNA and other small RNAs.,More than 10 Subunits to form RNA polymerase II,Have you ever wondered how your genome works?Well,thanks to scientists like R
14、oger Kornberg,awarded the 2006 Nobel prize for Chemistry,who has painstakingly studied the micromechanics of transcription,were getting a much clearer picture of what happens inside the nucleus,and how the billions of metres of DNA in your body are converted into RNA by an enzyme called RNA polymera
15、se II.-from the announcement of 2006 Nobel prize for chemistry,Animal RNA Polymerases,Animal DNA-dependent RNA Polymerases Class-amanitin sensitivity Major Products I Insensitive rRNA II Low Conc.(1-10 nM)hnRNA III High conc.tRNA,5S RNA and small RNAs,Amanita phalloides(the death cap),Structure of-a
16、manitin,Eukaryotic Transcription Initiation,Transcription initiation needs promoter and upstream regulatory regions.The cis-acting elements are the specific sequences on the DNA template that regulate the transcription of one or more genes.,Cis-acting element,RNA-pol does not bind the promoter direc
17、tly.RNA-pol II associates with six transcription factors,TFII A-TFII H.The trans-acting factors are the proteins that recognize and bind directly or indirectly cis-acting elements and regulate its activity.,Transcription factors,Enhancer,Enhancers Work Upstream,Downstream or in the Middle of a Gene
18、They also work forwards or backwardsPossible ways of working Different transcription factors Order of binding(differing concentrations)Affinity of transcription factors,TATA box is a septamer(TATAAAA)at-25 and is involved in positioning the enzyme for correct initiation.,CAAT box(CCAATCT)is at 75 an
19、d is recognized by a large group of transcription factors and plays a strong role in determining the efficiency of the promoter.,GC box is at-90 contains the sequence GGGCGG and is recognized by the factor SP1.,Elements combination in type II Promoters,Enhancer;Dehancer;Silencer;Upstream Activating
20、Sequences(UAS),TFIIA activates TBP by relieving the repression that is caused by the TAFs TFIIB binds adjacent to TBP and TATA boxTFIID is a complex protein containing a TATA-box binding protein and 8-10 TBP-associated factors(TAFs)TBP:TATA-binding protein TAFs:TBP-associated factors TFIIF consists
21、of two subunits.The larger subunit has an ATP-dependent DNA helicase activity and the small one contacts the core polymerase.TFIIE and TFIIH are required for promoter clearance to allow RNA polymerase to commence movement away from the promoter.,Class II transcription factors,RNA pol with transcript
22、ion factors form transcription initiation complex.TF II D is the only factor which can recognize specific sites.,TATA,TFF,A,B,Pre initiation complex,H,E,TBP,TAF,TFD-A-B-DNA complex,A,B,TATA,H,E,CTD tail of RNA pol II is phosphorylated by TFH,TBP,TAF,TBP,TAF,TBP,TAF,TBP,CTD（Carboxyl Terminal Domain)i
23、s repeated sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser,TFIIH has several activities,including an ATPase,a helicase,and a kinase activity that can phosphorylate the CTD tail of RNA polymerase II;it is also involved in repair of damage to DNA.,Most of the TFII factors are released before RNA polymerase II
24、 leaves the promoter.,Phosphorylation of the CTD by the kinase activity of TFIIH may be needed to release RNA polymerase to start transcription.,End of Initiation,Transcription unit for RNA polymerase I,Promoters in type III gene,upstream promoter(type 3)and internal promoter(type1,2)upstream promot
25、er:U6 snRNA Internal promoter:5S RNA and tRNA,Initiation in type 1 and 2 gene with polymerase III,tRNA,5S RNA,Initiation RNA pol I RNA pol III RNA pol II_ATP requirement no no yes_ A and B or TATA box core consensus sq.core element C box Inr _ CAAT box upstream element UCE GC box etc_ general TFs SL
26、1 TFIIIA B C various TFIIs_ upstream factors UBF various up-stream factors_,TBP is a universal factor,Transcriptional elongation,CTD phosphorylation status of RNA pol II,Steps leading to transcriptional activation,Promoter escape/clearance,Transition to elongation phase,What happens during transcrip
27、tional elongation?,Original contacts within pre-initiation complex abolished,Formation of new contacts with elongation factors,Phosphorylation of CTD,Change of RNA pol II to a ternary complex=high stability,CTD consists of heptad repeats of the consensus sequence:YSPTSPS,Promoter clearance:Ser#5 get
28、s phosphorylated,Transition to elongation:Ser#2 gets phosphorylated,Experimental evidence for elongation factors,Comparison of RNAPII elongation rate,in vitro:100-300 nt/min,frequent pauses,and sometimes full arrest,in vivo:1200-2000 nt/min,Why the discrepancy?,Use of pharmacological agents,DRB(5,6-
29、dichloro-1-D-ribofuranosylbenzimidazole,DRB,nucleotide-analogue,cause inhibition of hnRNA transcription by arresting RNA pol II in vivo,but not purified RNA pol II.Possible target?,These evidence suggest existence of factors that facilitate transcriptional elongation,RNA polymerase II often encounte
30、rs pauses&arrests,Arrest(irreversible backsliding 7-14 nts),Pause(back-tracking 2-4 nts),Function of elongation factors:minimize these pauses&arrests,HIV virus can transactivate by hijacking elongation machinery,HIV can bypass pre-initiation complex and head straight for elongation by hijacking RNA
31、pol II from host,P-TEFb phosphorylates RNA polI CTD,Tat:HIVs own elongation factor,Termination of Eukaryotic Transcription,The termination sequence is AATAAA followed by GT repeats.The termination is closely related to the post-transcriptional modification.,Type II genes:Transcription stops after AA
32、TAAA-Polyadenylation signal.Type I genes:3-4 consecutive TsType III genes:Stop after synthesis of serial Us.,Methodology in Gene Transcription,Reporter gene transcription Gel mobility shiftDNase Footprinting ChIPNuclear run-on transcription,Change promoter sequence Promoter elements are defined by m
33、utations and footprinting,How to determine promoter elements,Gel Mobility Shift,DNase Footprinting,Identification of Target Genes by ChIP Promoter Array,Chromatin Immunoprecipitation,Post-transcriptional Processing of RNA,Making ends of RNARNA splicing,Primary Transcript,Primary Transcript-the initi
34、al molecule of RNA produced-hnRNA（heterogenous nuclear RNA）In prokaryotes,DNA RNA protein in cytoplasm concurrently In eukaryotes nuclear RNA Cp RNA,Processing of eukaryotic pre-mRNA,Human dystrophin gene has 79 exons,spans over 2,300-Kb and requires over 16 hours to be transcribed!,For primary tran
35、scripts containing multiple exons and introns,splicing occurs before transcription of the gene is complete-co-transcriptional splicing.,Types of RNA processing,A)Cutting and trimming to generate ends:rRNA,tRNA and mRNAB)Covalent modification:Add a cap and a polyA tail to mRNAAdd a methyl group to 2-
36、OH of ribose in mRNA and rRNAExtensive changes of bases in tRNAC)Splicingpre-rRNA,pre-mRNA,pre-tRNA by different mechanisms.,The RNA Pol II CTD is required for the coupling of transcription with mRNA capping,polyadenylation and splicing,The coupling allows the processing factors to present at high l
37、ocal concentrations when splice sites and poly(A)signals are transcribed by Pol II,enhancing the rate and specificity of RNA processing.The association of splicing factors with phosphorylated CTD also stimulates Pol II elongation.Thus,a pre-mRNA is not synthesized unless the machinery for processing
38、 it is properly positioned.,Time course of RNA processing,5 and 3 ends of eukaryotic mRNA,Add a GMPMethylate it and1st few nucleotides,Cut the pre-mRNAand add As,5-UTR,3-UTR,Capping of pre-mRNAs,Cap=modified guanine nucleotideCapping=first mRNA processing event-occurs during transcriptionCTD recruit
39、s capping enzyme as soon as it is phosphorylatedPre-mRNA modified with 7-methyl-guanosine triphosphate(cap)when RNA is only 25-30 bp longCap structure is recognized by CBC（cap-binding complex）stablize the transcript prevent degradation by exonucleases stimulate splicing and processing,Sometimesmethy
40、lated,Sometimesmethylated,Capping of the 5 end of nascent RNA transcripts with m7G,Existing in a single complex,The cap is added after the nascent RNA molecules produced by RNA polymerase II reach a length of 25-30 nucleotides.Guanylyltransferase is recruited and activated through binding to the Ser
41、5-phosphorylated Pol II CTD.The methyl groups are derived from S-adenosylmethionine.Capping helps stabilize mRNA and enhances translation,splicing and export into the cytoplasm.,Consensus sequence for 3 process,AAUAAA:CPSF(cleavage and polyadenylation specificity factor)GU-rich sequence:CstF(cleavag
42、e stimulation factor F),Polyadenylation of mRNA at the 3 end,CPSF:cleavage and polyadenylation specificity factor.CStF:cleavage stimulatory factor.CFI&CFII:cleavage factor I&II.PAP:poly(A)polymerase.PABPII:poly(A)-binding protein II.,Poly(A)tail stabilizes mRNA and enhances translation and export in
43、to the cytoplasm.,RNA is cleaved 1035-nt 3 to A2UA3.,The binding of PAP prior to cleavage ensures that the free 3 end generated is rapidly polyadenylated.,PAP adds the first 12A residues to 3-OH slowly.,Binding of PABPII to the initial short poly(A)tail accelerates polyadenylation by PAP.,The polyad
44、enylation complex is associated with the CTD of Pol II following initiation.,Functions of 5 cap and 3 polyA,Need 5 cap for efficient translation:Eukaryotic translation initiation factor 4(eIF4)recognizes and binds to the cap as part of initiation.Both cap and polyA contribute to stability of mRNA:Mo
45、st mRNAs without a cap or polyA are degraded rapidly.Shortening of the polyA tail and decapping are part of one pathway for RNA degradation in yeast.,mRNA Half-life,t seconds if seldom needed t several cell generations(i.e.48-72 h)for houskeeping gene avg 3 h in eukaryotes avg 1.5 min in bacteria,Po
46、ly(A)+RNA can be separated from other RNAs by fractionation on Sepharose-oligo(dT).,Split gene and mRNA splicing,Background:Adenovirus has a DNA genome andmakes many mRNAs.Can we determine whichpart of the genome encodes for each mRNA bymaking a DNA:RNA hybrid?Experiment:Isolate Adenovirus genomic D
47、NA,isolate one adenovirus mRNA,hybridize and then look by EM at where the RNA hybridizes(binds)to the genomic DNA.Surprise:The RNA is generated from 4 different regions of the DNA!How can weexplain this?Splicing!,The discovery of split genes(1977)1993 Noble Prize in Medicine To Dr.Richard Robert and
48、 Dr.Phillip Sharp,The matured mRNAs are much shorter than the DNA templates.,DNA,mRNA,Exon and Intron,Exon is any segment of an interrupted gene that is represented in the mature RNA product.Intron is a segment of DNA that is transcribed,but removed from within the transcript by splicing together th
49、e sequences(exons)on either side of it.,Exons aresimilar in size,Introns are highlyvariable in size,GT-AG rule,GT-AG rule describes the presence of these constant dinucleotides at the first two and last two positions of introns of nuclear genes.Splice sites are the sequences immediately surrounding
50、the exon-intron boundaries Splicing junctions are recognized only in the correct pairwise combinations,The sequence of steps in the production of mature eukaryotic mRNA as shown for the chicken ovalbumin gene.,The consensus sequence at the exonintron junctions of vertebrate pre-mRNAs.,4 major types
