生化2014 c16 rna transcription2.ppt
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1、Biochemistry,Dept. of Biochemistry and Molecular Biology,Professor Wu Yaosheng 2014-06,2,Chapter 16,RNA Biosynthesis,3,Two kinds of RNA biosynthesis in organisms:,Transcription,RNA replication,3,4,Transcription-to synthesize RNA with DNA single strand as template,Introduction,1. Transcription is the
2、 first stage of the process of gene expression.,2. Transcription processes have to suffer strictly regulation to meet the need of development, morphogenesis and physiological functions of organisms .,3. The products of transcription are RNA,4,5,General picture of transcription,5,6,Main Contains, Ove
3、rview of Transcription, Transcription in Eukaryotes, Processing and degradation of Eukaryotic RNA, Transcription in Prokaryotes,6,7,Key Points, Transcription characteristics, Transcription system, RNA polymerases, Transcription initiation & stop, Postranscription modification,7,8,Section One,Templat
4、e and enzyme of prokaryotic transcription,9,Transcription,The process which RNA polymerase catalyzes the yield of RNA (tRNA, mRNA, rRNA ) with one of double strands of DNA as template, NTPs as precursors, in the light of the rule of complementary base pairing.,9,10,Precursors : NTP (ATP, UTP, GTP, C
5、TP) Template : DNA (one strand ) Enzyme: RNA polymerase, RNA-pol Other proteins (transcriptional factors et al ),The requirements of transcription,10,11,Comparison of Replication and Transcription,11,12,Only one strand of DNA molecule serves as template Asymmetric transcription No primer needed Init
6、iation at promoter site,1. General characters of transcription,12,13,DNA Transcription plot,5GCAGTACATGTC3 3c g t c a t g t a c a g5,DNA,5GCA GUA CAU GUC3,mRNA,N. Ala Val His Val .C,peptide,transcription,translation,Note:capital letters means the code strand, small letters means the template strand,
7、13,14,Some important concepts,DNA template,Template strand, antisense strand, Watson strand,Coding strand, sense strand, Crick strand,Structural gene,Asymmetric transcription,14,15,15,16,Asymmetry transcription,Structure gene,Template strand,5,3,5,3,Arrowhead means the direction of transcription,5,5
8、,5,Coding strand,16,17,For asymmetric transcription, there are two meanings:,(1) Only one strand of a gene can serve as template, the other which is complimentary to the template strand cant be transcribed.,(2) Not all the template strands of genes are found in the same strand on DNA molecule.,17,18
9、,The enzyme related to transcription is RNA polymerase, which is termed as DNA dependent RNA polymerase (DDRP) or RNA pol, or transcriptase.,2. DNA Dependent RNA Polymerase,It catalyzes the following reaction:,(NTP)n,pppN(pN)n-1 + (n-1)PPi,DNA template,RNA pol,Mg2+, Zn2+,18,19,DNA template strand,DN
10、A template strand,OH,OH,OH,OH,U,U,Transcription by RNA polymerase. In each step the incoming ribonucleotide selected is that which can base-pair with the next base of the DNA template strand.,direction,5,3,19,20,A 3-5phosphodiester bond is formed, extending the RNA chain by one nucleotide, and pyrop
11、hosphate is released. Overall the RNA molecule grows in a 53direction.,20,21,Prokaryotic RNA polymerase ( E. coli),21,22,only one RNA pol has been found: 2 (holoenzyme ) 2 ( core enzyme ),Core enzyme,Holoenzyme,RNA polymerase in prokaryotes,22,23,Holoenzyme: It can initiate transcription specificall
12、y at promoter sites and catalyze polymerization of two free NTPs. Core enzyme: It can not initiate transcription specifically at promoter sites but can catalyze RNA elongation.,Core enzyme: the RNA polymerase without the subunit is called core enzyme (2 ).,23,24,There are more than one kind of facto
13、r in prokaryotes, 70 carries out the promoter recognition process on their own, mainly responsible for the housekeeping gene expression., 32 is responsible for the heat-shock gene expression under some emergent cases.,Housekeeping genes are those that encode many proteins needed for routine cell fun
14、ctions and which are therefor expressed at low rates in all cells.,24,25,RNA polymerase binds to DNA at the Transcriptional start point,25,How many kinds of RNA polymerase have been found in prokaryotes?,26,Questions,1. The RNA polymerase that transcribe bacterial DNA is,A. multisubunit enzyme. B. m
15、onomeric and very large. C. multimeric and interchangeable. D. only active inside the cell. E. highly glycosylated in their active forms.,26,27,Questions,2. RNA chain elongation can be inhibited by,A. Repressors. B. Rifampicin (a Rifamycin derivative). C. Actinomycin D. D. RNases. E. 0.5 M NaCl.,27,
16、28,Section Two,Transcription in Prokaryotes,29,Initiation,Elongation,Termination,1. Proceeding of Transcription in Prokaryotes,29,30,1.1 Initiation of transcription,The transcriptional unit in prokaryotes is operon that consists of two regions on DNA,Operon,30,31,Regulation region,P,O,I,TTGACA,TATAA
17、TPu,Operon structure,Inhibitive protein,substrate,Structural gene region,31,32,During initiation, RNA polymerase recognizes promoter site, and then unwinds DNA locally to expose a single-stranded DNA template that can be transcribed.,Initiation of transcription,Recognition of promoter,Start of synth
18、esis,32,33,The steps of transcription initiation,(1) The sigma factor() in holoenzyme of RNA pol recognizes the promoter and let the whole enzyme to bind with the promoter sequence.,(2) To unwind the local region of promoter on DNA and to form a transcription initiation bubble .,(3) To form a initia
19、tion complex of transcription.,Holoenzyme- DNA-pppGpN-OH,33,34,34,35,Transcriptional start site,Transcriptional bubble,pppG-OH + pppN-OH pppGpN-OH + PPi,Holoenzyme of RNA pol,35,36,TATAAT,-10,RNA pol,36,37,-10 sequence: Located about 10 nucleotides upstream of where transcription will begin. -35 seq
20、uence: Located about 35 nucleotides upstream.,TTGACA,TATAAT,-35,-10,Transcriptional start site,5,By convention, the first nucleotide of the template DNA that is transcribed into RNA is denoted +1, the transcriptional start site.,+1,Two important sequence in prokaryotic promoters:,?,37,38,Consensus s
21、equence of promoter in E. coli,Pribnow box,Promoter,38,39,1.2 Elongation of transcription,The elongation phase of RNA synthesis, which begins after formation of the first bond, is carried out by core enzyme.,The transcriptional complex:,Core enzyme of RNA pol-DNA template-new RNA,39,40,Core enzyme(2
22、) moves along the gene, synthesizes a complementary RNA copy to the DNA template, using four ribonucleoside 5 triphosphates (ATP,CTP,GTP,UTP) as precursors.,-subunit dissociates from the enzyme, once transcription has been initiated .,3-OH at the end of the growing RNA chain attacks the phosphate gr
23、oups of the incoming ribonucleoside 5 triphosphate to form a 35 phosphodiester bond.,40,41,The complex of RNA polymerase, DNA template and new RNA transcript is called a transcription bubble. Because within it there is a region where the DNA double helix has opened up to allow transcription to occur
24、.,The RNA transcript forms a transient RNA-DNA hybrid helix with its template strand but then peels away from the DNA as transcription proceeds.,Transcription bubble,41,42,transcription bubble,RNA-pol (core enzyme) DNA RNA,42,43,Direction : RNA is synthesized in the 53direction. DNA template is read
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