1、Transcription and Post-transcription ModificationCentral dogmaWHICH 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 mak
2、e proteins.So which came first?RNA and RNA worldWalter Gilbert1980 Nobel Prize Origin-of-Life Theories 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 stages
3、: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 as
4、 the asymmetric transcription.Asymmetric transcriptionTemplateThe 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.Therefore,it i
5、s 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 ReplicationTranscriptionTemplateDouble strandsSingle strandSubstratedNTPNTPPrimer yesno Enzyme
6、DNA polymeraseRNA polymeraseProductdsDNAssRNABase pairA-T,G-CA-U,T-A,G-CDifferences between replication and transcription The whole genome of DNA needs to be replicated,but only small portion of genome is transcribed in response to the development requirement,physiological need and environmental cha
7、nges.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 Three phases:initiation,elongation,and termination.The prokaryotic RNA-pol can bind to the DNA template directly in the transcription process.The eu
8、karyotic RNA-pol requires co-factors to bind to the DNA template together in the transcription process.Transcription bubbleTranscription in prokaryotesTranscription Unit Optimal PromoterThe 35 sequence is used for initial recognition,and the 10 sequence is used for the melting reaction that converts
9、 a closed complex to an open complex.During transcription,the bubble is maintained within bacterial RNA polymerase,which unwinds and rewinds DNA,maintains the conditions of the partner and template DNA strands,and synthesizes RNA.Bacterial RNA Polymerases A single type of RNA polymerase is responsib
10、le 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 2,0005,000 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 t
11、arget of RifamycinHow many sigma factors exist in E.coli?How does transcription initiate?Four stages of TranscriptionTermination The terminator is in the transcript,not the DNA Forms a hairpin Self-complementary The hairpin structure is the signal for termination Rho()-dependent vs.-independent Intr
12、insic terminators-independentAn inverted repeat that allows a hairpin to form at the end of the transcriptsA string of Ts in the nontemplate strand that results in a string of weak rU-dA base pairs holding the transcript to the template strandRho factor pursues RNA polymerase along the RNA and can c
13、ause termination when it catches the enzyme pausing at a rho-dependent terminator.Termination of-dependentTranscription in EukaryotesRNA polymerases in Eukaryotes RNA polymerase I transcribes rRNA RNA polymerase II transcribes hnRNA（heterogeneous nuclear RNA）RNA polymerase III transcribes tRNA and o
14、ther small RNAs.More than 10 Subunits to form RNA polymerase II Have you ever wondered how your genome works?Well,thanks to scientists like Roger Kornberg,awarded the 2006 Nobel prize for Chemistry,who has painstakingly studied the micromechanics of transcription,were getting a much clearer picture
15、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 polymerase II.-from the announcement of 2006 Nobel prize for chemistry Animal RNA Polymerases Animal DNA-dependent RNA Polymerases Class -amanitin sensitivity Major Pr
16、oducts 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-amanitin Eukaryotic Transcription Initiation Transcription initiation needs promoter and upstream regulatory regions.The cis-acting elements are the specific se
17、quences on the DNA template that regulate the transcription of one or more genes.structural geneGCGCCAATTATAintronexonexonstartCAAT boxGCboxenhancercis-acting elementTATA box(Hogness box)Cis-acting element RNA-pol does not bind the promoter directly.RNA-pol II associates with six transcription facto
18、rs,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 EnhancerEnhancers Work Upstream,Downstream or in the Middle of a Gene They also work forwards or backwardsPossible ways of wor
19、king 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 and is recognized by a large group of transcription factors
20、 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 Sequences(UAS)TFIIA activates TBP by relieving the repressi
21、on 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 of two subunits.The larger subunit has an ATP-dependent DNA
22、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 factorspolTFHTAFTFFTAFTAFTFATFBTBPRNA pol with transcription factors form transcription init
23、iation complex.TF II D is the only factor which can recognize specific sites.TATA DNATATAPOL-TFFABPre initiation complexPOL-TFFHETBP TAFTFD-A-B-DNA complexABTATAHECTD-PCTD tail of RNA pol II is phosphorylated by TFHTBP TAFTBP TAFTBPTAFTBPCTD（Carboxyl Terminal Domain)is repeated sequence of Tyr-Ser-P
24、ro-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 leaves the promoter.Phosphorylat
25、ion 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 promoter:U6 snRNA Internal promoter:5S RN
26、A and tRNAInitiation in type 1 and 2 gene with polymerase III tRNA5S 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 SL1 TFIIIA B C various TFIIs_ upstream
27、factors UBF various up-stream factors_TBP is a universal factor Transcriptional elongationCTD phosphorylation status of RNA pol IISteps leading to transcriptional activation Promoter escape/clearanceTransition to elongation phaseWhat happens during transcriptional elongation?Original contacts within
28、 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 gets phosphorylated Transition to elongation:S
29、er#2 gets phosphorylatedExperimental 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/minWhy the discrepancy?Use of pharmacological agents DRB(5,6-dichloro-1-D-ribofuranosylbenzimidazole DRB,nu
30、cleotide-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 encounters pauses&arrests Arrest(irreversible backslidi
31、ng 7-14 nts)Pause(back-tracking 2-4 nts)Function of elongation factors:minimize these pauses&arrestsHIV virus can transactivate by hijacking elongation machineryHIV can bypass pre-initiation complex and head straight for elongation by hijacking RNA pol II from hostP-TEFb phosphorylates RNA polI CTDT
32、at:HIVs own elongation factorTermination 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 AATAAA-Polyadenylation signal.Type I genes:3-4 consecutiv
33、e Ts Type 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 mutations and footprintingHow to determine promoter elem
34、entsGel Mobility ShiftDNase Footprinting Identification of Target Genes by ChIP Promoter ArrayChromatin Immunoprecipitation Post-transcriptional Processing of RNAMaking ends of RNARNA splicingPrimary Transcript Primary Transcript-the initial molecule of RNA produced-hnRNA（heterogenous nuclear RNA）In
35、 prokaryotes,DNA RNA protein in cytoplasm concurrently In eukaryotes nuclear RNA Cp RNA Processing of eukaryotic pre-mRNAHuman dystrophin gene has 79 exons,spans over 2,300-Kb and requires over 16 hours to be transcribed!For primary transcripts containing multiple exons and introns,splicing occurs b
36、efore transcription of the gene is complete-co-transcriptional splicing.Types of RNA processingA)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-OH of ribose in mRNA and rRNAExtensive changes of bases in tRNAC)
37、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 1.The coupling allows the processing factors to present at high local concentrations when splice sites and poly(A)signals are tra
38、nscribed by Pol II,enhancing the rate and specificity of RNA processing.2.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 it is properly positioned.Time course of RNA processing 5 and
39、 3 ends of eukaryotic mRNAAdd a GMPMethylate it and1st few nucleotidesCut the pre-mRNAand add As5-UTR3-UTRCapping of pre-mRNAs Cap=modified guanine nucleotide Capping=first mRNA processing event-occurs during transcription CTD recruits capping enzyme as soon as it is phosphorylated Pre-mRNA modified
40、 with 7-methyl-guanosine triphosphate(cap)when RNA is only 25-30 bp long Cap structure is recognized by CBC（cap-binding complex）stablize the transcript prevent degradation by exonucleases stimulate splicing and processingSometimesmethylatedSometimesmethylatedCapping of the 5 end of nascent RNA trans
41、cripts with m7GExisting 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 Ser5-phosphorylated Pol II CTD.The methyl groups are derived from S-ade
42、nosylmethionine.Capping helps stabilize mRNA and enhances translation,splicing and export into the cytoplasm.Consensus sequence for 3 processAAUAAA:CPSF(cleavage and polyadenylation specificity factor)GU-rich sequence:CstF(cleavage stimulation factor F)Polyadenylation of mRNA at the 3 endCPSF:cleava
43、ge 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 into the cytoplasm.RNA is cleaved 1035-nt 3 to A2UA3.The binding of PAP pri
44、or 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 polyadenylation complex is associated with the CTD of Pol II following initiation.Fu
45、nctions 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:Most mRNAs without a cap or polyA are degraded rapidly.Shortening of the polyA ta
46、il 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 Poly(A)+RNA can be separated from other RNAs by fractionation on Sepharose-oligo(d
47、T).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 DNA,isolate one adenovirus mRNA,hybridize and then look by EM at where the RNA hyb
48、ridizes(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 Dr.Phillip SharpThe matured mRNAs are much shorter than the DNA templates.DNAmRNA
49、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 the sequences(exons)on either side of it.Exons aresimilar in sizeIntrons are highlyvaria
50、ble in sizeGT-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 the exon-intron boundaries Splicing junctions are recognized only in the correct pairwise
