1、RNA Biosynthesis(Transcription)1复制和转录过程有什么相似之处？复制和转录过程有什么相似之处？又各有什么特点？又各有什么特点？2. 试述真核细胞试述真核细胞RNA转录后的加工修转录后的加工修饰饰 Concept The process of copying DNA to RNA by an enzyme called RNA polymerase (RNAP) The transfer of genetic information from DNA into RNAtranscriptionRNADNA ContentlTemplates and Enzymesl
2、The Process of TranscriptionlPost-transcriptional ModificationThe difference between replication and transcriptionSubstances for transcriptionSubstrates: NTP (ATP, UTP, GTP, CTP) Template: DNAEnzyme: RNA polymerase( RNA-pol)Other protein factorsTemplates and EnzymesSection ITemplate for transcriptio
3、n Structural gene : a DNA fragment coding for any RNA or protein product other than a regulatory element Template strand (sense strand or Watson strand) : the strand of DNA that the RNA polymerase uses as a template to produce complementary mRNA Coding strand (antisense strand or Crick strand) :the
4、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 33 c g t g a t g t a c a g 55GCAGUACAUGUC 3NAla Val His Val CCoding standTemplate strandmRNAProteintranscriptiontranslation5 5 3 3 3 3 5 5 Templ
5、ate strandCoding strandCoding strandTemplate strandStructural geneDirection of transcriptionDirection of transcriptionAsymmetric 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 strandRNA
6、polymeraseProkaryotic RNA polymerasecore enzymeholoenzyme The binding site of RNApol holoenzyme on the transcription initiation regionEukaryotic RNA polymerase种种 类类对对 鹅鹅 膏膏 蕈蕈 碱碱的的 反反 应应45S-rRNAhnRNA5S-rRNAtRNAsnRNA耐耐 受受极极 敏敏 感感中中 度度 敏敏 感感转转 录录 产产 物物A site for RNA pol to identify and bind on the tem
7、plate Operon: a unit of transcription in prokaryotes including one or more structural genes and upstream regulon 5 3 3 5 Structural generegulonRNA-polpromoter : the site on the template DNA for RNA pol bindingRNA polymerase protectionassay目目 录录Transcription initiationT T G A C AA A C T G T-35 region
8、(Pribnow box)T A T A A T Pu A T A T T A Py-10 region1-30-5010-10-40-205 3 3 5 Prokaryotic promote conservative sequencerecognition site 5 5 RNA pol protection regionStructural gene3 3 TATA bax CAAT bax GC baxenhancerCis-actin elementStructural gene-GCGC-CAAT-TATATranscription initiationEukaryotic pr
9、omote conservative sequenceThe Process of TranscriptionSection IIInitiationTwo 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 mRNAProkaryotic transcription The
10、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 DNA1st poly reaction catalyzed by RNA pol and transcription initiation complex is formedRNApol ( 2) - DNA - pppGpN- OH 3 transcrip
11、tion initiation complex5 -pppG -OH + NTP 5 -pppGpN - OH 3 + ppiInitiationElongation 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 + PPitranscription bubble：RNA-pol （Core enzy
12、me ） DA RNA目目 录录目目 录录5 3 DNAFeather phenomena during prokaryotic transcriptionribosomeRNARNA pollRho () factor dependent transcription terminationlRho-independent transcription terminationTerminationThe process of the RNA polymerase stopping on DNA template and the dissociation of the RNA from the t
13、ranscription complexClassificationA T PRho () factor dependent transcription terminationTermination independent on Rho factorThe 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 R
14、NAP from the DNA template5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU. 3 5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU. 3 RNA 5 TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT. 3 DNA UUUU. UUUU.5UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU. 3stem-loop/ha
15、irpin structureMechanism involved in the termination by stem loopallosteric change of RNA poly for the stop of transcription dissociation of transcription complex for the release of RNA5 pppG5 3 3 5 RNA-polEukaryotic transcription initiationinitiationA variety of upstream control elements existRNA-p
16、ol does not directly bind to the template in the initiationMuch more complicated than prokaryotic transcriptionTranscriptionInitiation n siteTATA boxCAAT boxGC box enhancercis-acting elementThe upstream region from the transcription initiationAATAAAExcision andadding tailTranscriptionTermination sit
17、eModification siteexonexonTranslation initiation siteintronOCT-1 OCT-1：ATTTGCAT octamerTranscription factorTrans-acting factors :The proteins with ability to recognize and bind directly or indirectly to the upstream control element of DNAHundreds factors have been reportedTranscriptional factors (TF
18、): Among the trans-acting factors, the proteins with ability to bind directly or indirectly to RNA pol参与参与RNA-pol转录的转录的TF 蛋蛋白白激激酶酶活活性性，使使CTD磷磷酸酸化化TFHATPase57（ ） 34（ ）TFE解解螺螺旋旋酶酶30，74TFF促促进进RNA-pol结结合合及及作作为为其其他他因因子子结结合合的的桥桥梁梁33TFB稳稳定定TFD-DNA复复合合物物12，19，35TFA辅辅助助TBP-DNA结结合合TAF*结结合合TATA盒盒TBP* 38TFD功功能能
19、亚亚基基组组成成，分分子子量量(kD)转转录录因因子子蛋蛋白白激激酶酶活活性性，使使CTD磷磷酸酸化化TFHATPase57（ ） 34（ ）TFE解解螺螺旋旋酶酶30，74TFF促促进进RNA-pol结结合合及及作作为为其其他他因因子子结结合合的的桥桥梁梁33TFB稳稳定定TFD-DNA复复合合物物12，19，35TFA辅辅助助TBP-DNA结结合合TAF*结结合合TATA盒盒TBP* 38TFD功功能能亚亚基基组组成成，分分子子量量(kD)转转录录因因子子Pre-initiation complex ( PIC) Eukaryotic transcription initiation re
20、quire a variety of transcription factor because of the indirectly binding of Eukaryotic RNA-pol to DNA POL-TFFABPIC transcription catalyzed by RNA-Pol POL-TFFHETBPTAFTFD-A-B-DNA complexTATAABTBPTAFTATAHECTD-PAfter PIC assemble，CTD phosphorylation by TFH Piecing theory The eukaryotic transcription re
21、quires 3 to 5 TF binding together and becoming the complex for the activation of specific gene transcriptionElongationSimilar to prokaryotic transcription elongation. synchronous transcription and translation is not observed because of segregation of nuclear membrane The forward moving process of RN
22、A-pol encountering nucleosomeThe translocation and dissociation of nucleosomes observed during the transcriptionRNA-PolRNA-PolRNA-PolnucleosomeNucleosome translocation during elongationDirection of transcription5 -AAUAAA-5 -AAUAAA-nuclease-GUGUGUGRNA-polAATAAA GTGTGTGModification site fortranscripti
23、on termination5 5 3 3 3 adding tailAAAAAAA 3 mRNATerminationassociated with post-transcriptional modificationPost-transcriptional modificationSction IIIConceptIn eukaryotic cells, a genetic process for the conversion of a primary transcript RNA to mature RNAMajor modificationssplicingcleavagemodific
24、ationadditionEukaryotic mRNA processingModifications to the “head (5) and “tail (3) ends of the mRNA 5 capping (m7GpppGp ) 3 polyadenylation (poly A tail)5 cap structure5 pppGp5 GpppGppppG ppiGMPtransferase5 m7GpppGpmethyltransferase SAMThe formation of 5 cap 5 ppGpphosphatase PimRNA splicing hetero
25、-nuclear RNAa( hnRNA) : the primary mRNA within nucleus snRNA: a class of small RNA molecules within the nucleus Proteins within Nuclear small nuclear ribonucleoproteins （splicesome）snRNAa strand of DNA containing both introns and exons. Most advanced eukaryotes have interrupted genes and introns ar
26、e longer than exons. The resulting gene is much longer than its coding regionInterrupted gene CABDCoding region A、B、C、DNon-coding regionExon 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 precurso
27、r mRNA (pre-mRNA) and removed by a process called splicing during the processing to mature RNAChicken Ovalbumin Gene hnRNAModifications to the front and back endshnRNA splicingMature mRNAChicken Ovalbumin Gene and its Transcription and Post-transcriptional Modification 目目 录录Electron-microscopic pict
28、ure of the mature mRNA and DNA hybridization of Chicken Ovalbumin DNAmRNA目目 录录Classification of introns Four classes: according to gene types and splicing methodsI：mainly found in mitochondria, chloroplast, and certain low-level eukaryotic rRNA genesII： found in mitochondria, chloroplast, in mRNA ge
29、nesIII： frequently found in the splicing after loop formation ，in most mRNA genesIV：tRNA genes and primary transcription production intron ，splicing requiring enzyme and ATPmRNA splicing to remove the introns of hnRNA and ligate the exonsSplicesome is formed by ligation of snRNP and hnRNA 目目 录录UACUA
30、CA - AGUGU4U5U6E1E2U1U2UACUACA - AGUGU6E1E2U1、U4、U5pG-OH(ppG-OH, pppG-OH)U-OHGpUpGpA1ST transesterification reaction 2nd transesterification reaction UpAGpUExon 1IntronExon 2G-OHUpUpGpA剪接剪接过程的过程的二次转二次转酯反应酯反应 Differential RNA processing: RNA editing shows that the coding sequence of genes has a varie
31、ty of differential function after post-transcription processingmRNA editing Human apo B gene mRNA（14500 Nucleotides）liverapo B100（MW 500 000）intestinal cells apo B48（MW 240 000）mRNA editingPost-transcriptional process of tRNAtRNA precursorprecursorRNA pol TGGCNNAGTGCGGTTCGANNCCDNA目目 录录RNAase P、endon
32、uclease目目 录录tRNA nucleotide transferase 、ligaseATPADP目目 录录Base modificationsReduced reactione.g.：U DHU Translocation within nucleotidese.g.：U Deamination reaction e.g.：A I e.g.：A AmMethylation（1 1）（1 1）（3 3）（2 2）（4 4）目目 录录transcription45S - rRNAsplice18S - rRNA5.8S and 28S-rRNArDNAintronintron28S5.8
33、S18SPost-transcriptional process of rRNAan RNA molecule that catalyzes a chemical reactionRibozymeSecondary structure of Tetrahymena rRNA intronSelf-splicing rRNA of Tetrahymena5 -terminal sequence hammerhead structure The most simple secondary structure of ribozyme substrate Around 60 nucleotides W
34、ith 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-splicingSignificance of ribozyme researchlThe important supplementary to the central dogmalThe challenge to traditional enzy
35、mology lDesign a ribozyme according to the native ribozyme structureThe designed ribozyme Synthetic nucleic acids shown with thick lines Native nucleic acids shown with thin lines consensus sequence shown with Spliced site shown by arrow目目 录录AppendixGOH3 G5 OH5 3 GOH414G 3995 3 3955 3 E1E2ISelf-splicing RNA of Tetrahymena5 3 L19RNAFragment with catalytic activity
