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第 32 章 RNA 的生物合成与加工. 转录产物有 mRNA 、 tRNA 、 rRNA 。. 原核细胞的 mRNA 为单顺反子或多顺反子。. 真核细胞的 mRNA 为单顺反子。. 有义链,编码链;反义链,模板链. E.coli 的 RNA 聚合酶中, α 2 ββ ′ 称为核心酶。. σ 因子辨认模板 DNA 的起始位点。. 基因的转录 – mRNA 的合成. 基因转录是以 DNA 为模板合成与其碱基顺序互补的 mRNA 的过程。 - PowerPoint PPT Presentation
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32 RNAmRNAtRNArRNAmRNAmRNAE.coli RNA2DNA
mRNADNAmRNADNARNAmRNAmRNA
D. S. DNA DNARNA DNA3 5, DNARNA5 3.DNA(DNA)3----TACTCAT----5RNA 5----AUGAGUA----35---ATGAGTA----3Non-template (sense strand)template (antisense strand)
DNA RNApromotion, elongation, termination promoterterminator transcriptional unit polycistron in operon +1 monocistron, No operon
mRNA
RNA RNA5335 DNA DNA
DNADNADNARNA
DRNARNA-RNA
RNA (transcription)DNADNARNA
553355DNARNADNA(DNA
RNADNARNARNARNA RNA
RNADNA3'5'RNA5'3'
RNADNADNA
RNA ATPGTPCTPUTP DNA
RNADNARNADNA5'3'RNA
RNA2' 2'RNA
' DNA
RNARifampinRNA
RNA-1012
-
RNA pol rRNA
RNA pol HnRNA
RNA pol tRNA
snRNA5S rRNA
RNA
150kdRNARNA 2nusA69kdRNARNA
NusA protein ; 69 Kd, acid protein RNApol-attaching factor Impel RNApol. pausing in terminator for 1-15 and waiting for Rho factor to stop transcription of RNA (antitermination N protein utilization substance)
CAPcAMPCRP23kdcAMPRNA
RNA RNA -35TTGACA RNA-10TATAATPribnow DNA
RNADNApromoter) pppGpppA
Core promoter region including -35 (R)-10 (B)+1 (I)RNA
l Sextama Box Pribnow Box 17bp, RNApoll Sextama Box or Pribnow Box mut. 17 bpup mutation17 bpdown mutation17bp17bpSextama Box and Pribnow Box mut. 100X 1000X
(Re-usable) Holo-enzymeSextama Box, RNApol DNA107/mol R, B site (1014/mol)RNA
Promoterfactor strong promoter & weak promoter -35-10 ( ) -35, -10 RNApol factor is responsible for recognition of consensus sequence of promoter and only required for initiation.
TAHognessTATA CAATGC
Promoter for basic transcription ( class II recognized by RNA polymerase II ) l Cap site ; initiation point (+1) Capping m7GpppA/G------70 ---AUG---(in mRNA)
Promoter for basic transcription l TATA box / Hogness box / Goldberg-Hogness box (-30) Rich AT and rich GC flanked rich GC------T A T A A (T) A A (T)------rich GC 82 97 93 85 63 (37) 83 50 (37)
Promoter for basic transcription l GC island (C no methylated) and CAAT box (CAT box UPE) (-70) GCGC-----GGC(T)CAATCT---- Response to effect of transcription Range 30 bp
l Promoter functions CAAT box (modulator) controlling transcriptional efficiency (cause GC island) TATA box (selector) controlling starting point and efficiency I site (initiator)determination initiation point and capping of mRNA
RNAtrans-criptional factor, TF) TFATFBTFDTFETFFTF-I
Cis-factor for inducible expression HSE ( Heat Shock Element )GRE ( Glucocoricoid Response Element ) MRE ( Metal Response Element )TSE ( Tissue Special Element )l Inducible expressed by environment Ig ATGGAAAT + Oct-2 factor B GH ATGAATAT + Pit-1 factor
l EnhancerEnhance expression Basic expressionPosition not be fixed isolated region Bi-directional element Mono-directional element EnhancerPromoterChambon discovered the first enhancer inthe 5'-flanking region of the SV40 early gene.
Enhancer ----Enhancer (Enhancer Element) ----Enhancer Element , (Enhanson ----Enhanson(cis-factor)(trans-factor)
e.g. SV40 Enhancer (-179~ -250)
Silencer Mating type (MAT) of yeast
Silencer can act at a distance (at least 1 kb away) to modulate transcription somehow cause the chromatin to coil up into a condensed Inaccessible Inactive form thereby preventing transcription of neighboring genes
TFA TFD TFB pol TFD TATA TFE ATPase TFF RNA
trans-factor Trans-factor for basic transcription l TF (I, II, III) Transcriptional Factorl TBP (TATA box Binding Protein)l TAF (TBP Associate Factor)l RAP (RNApol. Associate protein)RNApol
RNApol II (B); ---For pre-mRNA transcription ---Located in nucleoplasm ---L maximum subunit 240 kd & have specific COOH-end named CTD ( Carboxyl Terminal Domain ) only in RNApol II ---CTD-end; 7aa repeats & high frequency phosphorylation TyrSerPProThrPSerPProSerP
Yeast 26XDrosophila 44X repeat unit of 7 aa / in CTDRat & Human 52X ---CTDSer,Thr L 3Subforms IIO (240kd) IIA (220kd) IIB (180kd) CTD over-phosphorylated 10X IIA Non-physiological form Initially binds to promoter
(TBP); needed for RNApol I, II, III Very high conserved C-end domain of 180 aa Binds with DNA in minor groove & wilds it Determination initiation starting site Control UPE effect for basic transcriptionTFIID; A sort of protein complex (TBP & > 8 TAF)
Promoter clearance Transcription startingF
Wilds minor groove pre-TIC Basic TIC conclusion
Promoter clearance Transcription startingcomplete TIC
Different acidic activation domain may regulation different cis-factor targets cis-factor positive control system
activator signalbinding DNA
lCys/Cys, Cys/His (Zinc finger)DNA-binding domainl Helix()-turn-Helix() (HTH) lHomeodomains (HDs 60aa with HTH)
9 direct repeats 30aa / copy
2Cys / 2His or 2Cys / 2Cys types
2Cys (2C) ~ Zn ~ 2His (2C) to form finger of 1-helix & 1-sheetZinc finger
Aaron Klug (1985)TFIIIA predict the Zinc finger structureTFIIIA three fingers lining up the major groove of DNA which sequence GCGTGGGCGCarl Pabo obtained the structure of complex between TFIIIA and DNA using X-ray crystallography
RNAATPGTP3',5'-
DNARNA
RNA1DNAGCATRNAURNARNA2()RNA
RNA mRNA 1(adding cap)mRNA5'-m7GTPHnRNA5'-GpppNG
RNAmRNAmRNARNA5 3polyA
RNASAMA3AAUAAARNA
2(adding tail)3'-polyApolyAmRNA
pre-RNA tailing A poly(A) tail (50-200) be added at -20 Nt tailing signal (AAUAAA) from 3-end of Pre-RNA
3splicing)HnRNAsnRNA
RNAG
4
tRNA
mRNA
tRNA
rRNA
rRNA
RNAUgRNARNARNAtRNAmRNA
Editing /AUG, UAA, UAG, UGA codon mRNA editing DNA DNA abbreviated cryptic gene, cryptogene
Editing