RNA Biosynthesis（transcription）RNA的生物合成课件.ppt

RNA Biosynthesis(Transcription),1复制和转录过程有什么相似之处？又各有什么特点？2.试述真核细胞RNA转录后的加工修饰,Concept The process of copying DNA to RNA by an enzyme called RNA polymerase(RNAP)The transfer of genetic information from DNA into RNA,transcription,Content,Templates and EnzymesThe Process of TranscriptionPost-transcriptional Modification,The difference between replication and transcription,Substances for transcription,Substrates:NTP(ATP,UTP,GTP,CTP)Template:DNAEnzyme:RNA polymerase(RNA-pol)Other protein factors,Templates and Enzymes,Section I,Template for transcription,Structural gene:a DNA fragment coding for any RNA or protein product other than a regulatory elementTemplate strand(sense strand or Watson strand):the strand of DNA that the RNA polymerase uses as a template to produce complementary mRNACoding strand(antisense strand or Crick strand):the strand that is not used as the templateit has the same base sequence as the RNA transcript produced(although with thymine replaced by uracil),5GCAGTACATGTC 3,3 c g t g a t g t a c a g 5,5GCAGUACAUGUC 3,NAla Val His Val C,Coding stand,Template strand,mRNA,Protein,transcription,translation,53,35,Template strand,Coding strand,Coding strand,Template strand,Structural gene,Asymmetric transcription,certain region of only one strand of DNA serves as a template for transcription The template stand is not always restricted to the same single DNA strand,RNA polymerase,Prokaryotic RNA polymerase,core enzyme,holoenzyme,The binding site of RNApol holoenzyme on the transcription initiation region,Eukaryotic RNA polymerase,A site for RNA pol to identify and bind on the template,Operon:a unit of transcription in prokaryotes including one or more structural genes and upstream regulon,promoter:the site on the template DNA for RNA pol binding,RNA polymerase protectionassay,目 录,Transcription initiation,T T G A C AA A C T G T,-35 region,(Pribnow box),T A T A A T Pu A T A T T A Py,-10 region,Prokaryotic promote conservative sequence,recognition site,Structural gene,-GCGC-CAAT-TATA,Eukaryotic promote conservative sequence,The Process of Transcription,Section II,Initiation,Two issues required to be addressed the binding of RNA polymerase to the initial region of the transcription template preciselyonly one strand of DNA,the template strand,is used to produce mRNA,Prokaryotic transcription,The DNA is unwound and becomes single-stranded in the vicinity of the initiation site(defined as+1).,RNA pol holoenzyme(2)specifically binding to the promoter region on DNA,1st poly reaction catalyzed by RNA pol and transcription initiation complex is formed,RNApol(2)-DNA-pppGpN-OH 3,transcription initiation complex,5-pppG-OH+NTP 5-pppGpN-OH 3+ppi,Initiation,Elongation,subunit dissociates from the holoenzyme，and elongation proceeds,with the effect of the core enzyme，NTP maintain polymerization，RNA strand elongated,(NMP)n+NTP(NMP)n+1+PPi,transcription bubble：,RNA-pol（Core enzyme）DA RNA,目 录,目 录,5,3,DNA,Feather phenomena during prokaryotic transcription,ribosome,RNA,RNA pol,Rho()factor dependent transcription terminationRho-independent transcription termination,Termination,The process of the RNA polymerase stopping on DNA template and the dissociation of the RNA from the transcription complex,Classification,A T P,Rho()factor dependent transcription termination,Termination independent on Rho factor,The terminator sequence within the RNA for the stop of RNA polymerase.The terminator sequence usually forms a stem-loop hairpin structure leading to the dissociation of the RNAP from the DNA template,5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU.3,5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU.3,RNA,5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT.3,DNA,5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU.3,stem-loop/hairpin structure,Mechanism involved in the termination by stem loop,allosteric change of RNA poly for the stop of transcription dissociation of transcription complex for the release of RNA,Eukaryotic transcription initiation,initiation,A variety of upstream control elements existRNA-pol does not directly bind to the template in the initiationMuch more complicated than prokaryotic transcription,TranscriptionInitiation n site,TATA box,CAAT box,GC box,enhancer,cis-acting element,The upstream region from the transcription initiation,AATAAA,Excision andadding tail,TranscriptionTermination site,Modification site,exon,Translation initiation site,intron,OCT-1,OCT-1：ATTTGCAT octamer,Transcription factor,Trans-acting factors:The proteins with ability to recognize and bind directly or indirectly to the upstream control element of DNAHundreds factors have been reported,Transcriptional factors(TF):Among the trans-acting factors,the proteins with ability to bind directly or indirectly to RNA pol,参与RNA-pol转录的TF,Pre-initiation complex(PIC),Eukaryotic transcription initiation require a variety of transcription factor because of the indirectly binding of Eukaryotic RNA-pol to DNA,TFF,A,B,PIC transcription catalyzed by RNA-Pol,H,E,TBP,TAF,TFD-A-B-DNA complex,TATA,A,B,TBP,TAF,TATA,H,E,After PIC assemble，CTD phosphorylation by TFH,Piecing theory,The eukaryotic transcription requires 3 to 5 TF binding together and becoming the complex for the activation of specific gene transcription,Elongation,Similar to prokaryotic transcription elongation.synchronous transcription and translation is not observed because of segregation of nuclear membrane,The forward moving process of RNA-pol encountering nucleosome,The translocation and dissociation of nucleosomes observed during the transcription,RNA-Pol,RNA-Pol,RNA-Pol,nucleosome,Nucleosome translocation during elongation,Direction of transcription,5-AAUAAA-,5-AAUAAA-,nuclease,-GUGUGUG,RNA-pol,AATAAA GTGTGTG,Modification site fortranscription termination,5,5,3,3,3 adding tail,AAAAAAA 3 mRNA,Termination,associated with post-transcriptional modification,Post-transcriptional modification,Sction III,ConceptIn eukaryotic cells,a genetic process for the conversion of a primary transcript RNA to mature RNA,Major modifications,splicing,cleavage,modification,addition,Eukaryotic mRNA processing,Modifications to the“head(5)and“tail(3)ends of the mRNA,5 capping(m7GpppGp)3 polyadenylation(poly A tail),5 cap structure,5 pppGp,The formation of 5 cap,mRNA splicing,hetero-nuclear RNAa(hnRNA):the primary mRNA within nucleus snRNA:a class of small RNA molecules within the nucleus,a strand of DNA containing both introns and exons.Most advanced eukaryotes have interrupted genes and introns are longer than exons.The resulting gene is much longer than its coding region,Interrupted gene,Coding region A、B、C、D,Exon and intron,exon the regions of a gene that are represented in mature form of the messenger RNA(mRNA)intronThe sections of a gene that are present in precursor mRNA(pre-mRNA)and removed by a process called splicing during the processing to mature RNA,Chicken Ovalbumin Gene,hnRNA,Modifications to the front and back ends,hnRNA splicing,Mature mRNA,Chicken Ovalbumin Gene and its Transcription and Post-transcriptional Modification,目 录,Electron-microscopic picture of the mature mRNA and DNA hybridization of Chicken Ovalbumin,DNA,mRNA,目 录,Classification of introns,Four classes:according to gene types and splicing methods,I：mainly found in mitochondria,chloroplast,and certain low-level eukaryotic rRNA genesII：found in mitochondria,chloroplast,in mRNA genesIII：frequently found in the splicing after loop formation，in most mRNA genesIV：tRNA genes and primary transcription production intron，splicing requiring enzyme and ATP,mRNA splicing,to remove the introns of hnRNA and ligate the exons,Splicesome is formed by ligation of snRNP and hnRNA,目 录,pG-OH(ppG-OH,pppG-OH),剪接过程的二次转酯反应,Differential RNA processing:RNA editing shows that the coding sequence of genes has a variety of differential function after post-transcription processing,mRNA editing,Post-transcriptional process of tRNA,tRNA precursor,目 录,目 录,tRNA nucleotide transferase、ligase,ATP,ADP,目 录,Base modifications,目 录,Post-transcriptional process of rRNA,an RNA molecule that catalyzes a chemical reaction,Ribozyme,Secondary structure of Tetrahymena rRNA intron,Self-splicing rRNA of Tetrahymena,5-terminal sequence,hammerhead structure The most simple secondary structure of ribozyme,substrate,Around 60 nucleotidesWith catalytic part and substrate in the same molecular hammerhead structure consisted of catalytic part and substrate,Apart from rRNA，the process of tRNA、mRNA also exists Self-splicing,Significance of ribozyme research,The important supplementary to the central dogmaThe challenge to traditional enzymology Design a ribozyme according to the native ribozyme structure,The designed ribozyme,Synthetic nucleic acids shown with thick linesNative nucleic acids shown with thin linesconsensus sequence shown withSpliced site shown by arrow,目 录,Appendix,Self-splicing RNA of Tetrahymena,