分子生物学第四章.ppt
基因的表达 Gene expression,第4章 RNA转录(RNA transcription),4.1 基本概念,4.2 原核生物RNA转录启动子,4.3 真核生物RNA转录启动子,4.4 Transcription Elongation,4.5 Transcription termination,4.6 Anti-termination in Rho-dependent terminator of Prokaryotes,4.7 Pre-RNA processing in Eukaryotes,4.1基本概念,基因表达的第一步,以D.S.DNA中的一条单链作为转录的模板,在依赖DNA的RNA聚合酶的作用下,模板单链 DNA的极性方向为3 5,而非模板单链 DNA的极性方向与RNA链相同，均为5 3.,DNA,(书写DNA序列时，仅写非模板序列，可不注明极性方向),3-TACTCAT-5,RNA 5-AUGAGUA-3,5-ATGAGTA-3,Non-template(sense strand),template(antisense strand),Jacob.Monod nonspecialized ribosomes that translate unstable RNAs called messengers(messenger RNA,mRNA)messengers are independent RNAs bring genetic information from the genes to ribosomes The music(polypeptide)depends on the tape(mRNA),not the player(ribosome),4.2 RNA Transcription promotion in prokaryotes,4.2.1 Promoter 的结构与功能(Prok.E.coli),a)promoter 由两个重要部分组成,-70-40 CAPcAMP binding site,-35-10 RNApol.binding site,基因表达调控的正控制位点,CAP:,cAMP Acceptor Protein,(环化AMP受体蛋白),b)CAP-cAMP binding site(Activator region,AR),ARI+CAP-cAMP,AR I：,CAP-cAMP 的强结合位点（-70-50）,cooperative effect,提高ARII 的结合效率,-70,AR I,-40,AR II,-50,AR II+CAP-cAMP 复合体 促使Sextama Box 附近GC岛区的双螺旋结构稳定性降低，Pribnow Box 的解链温度降低，利于转录启动,ARI ARII GC Island Sextama Pribnow,AR II+CAP-cAMP,promotion,RNApol.into Sextama Box,into Pribnow Box,starting transcription,RNApol,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.,14/16 down mutation,-Clac TATGTT,A A,LRNA-trp TTAACT,C,Lac-p115 TATTGT,A,-CiR TACACT,C,G,C,2/16 up mutation,Pribnow Box 中A/TT/A,改变了碱基堆积状况 RNApol.与模板链的结合效率，转录效率上升（上升突变）,Pribnow Box 含有G/C,site II 必须有 CAP-cAMP,以改变双螺旋体的稳定性 A/T G/C 双螺旋体稳定性 加强,转录率下降(下降突变),Sigma factor controls binding to DNA,Sigma factor is the subunit of bacterial RNA polymerase needed for initiationA major influence on selection of binding sites(promoters),RNA polymerase passes through several steps prior to elongation.A closed(means DNA remains duplex)binary complex is converted to an open(melting a short of region of DNA)form(tight binding)and then into a ternary complex(RNA,DNA and Enzyme).,RNA polymerase initially contacts the region from-55 to+20.When sigma dissociates,the core enzyme contracts to-30;when the enzyme moves a few base pairs,it becomes more compactly organized into the general elongation complex.,Core enzyme and holoenzyme are distributed on DNA,and very little RNA polymerase is free.,Excess core enzyme exists largely as closed loose complexes and free core enzyme is very little;One third are holoenzyme and distributed between loose complexes at nonspecific sites and binary complexes(mostly closed)at promoters;About half are core enzymes engaged in transcription;Free holoenzymes is small(?),How does RNA polymerase find target promoters so rapidly on DNA?,Sigma factor and core enzyme recycle at different points in transcription.,因子：,促进RNA pol+NTP RNA elongation,完成 NMP之间的磷酸脂键的连接,Editing,与 Rho()因子竞争RNA 3-end,构成Holo Enzyme后，因子含有 两个位点,I site(Rif S);,E site(Rif R);,要求高浓度的ATP or GTP,专一性地结合ATP or GTP,对 NTP 非专一性结合,Rifampin是RNA合成起始的抑制剂,因子：,强碱性亚基,促使RNA pol与非模板链（sense strand）强结合,受K酶抑制,Holo Enzyme 含有五个功能位点,sense strand DNA binding point()DNA/RNA hybrid site（形成磷酸二酯 键）()D.S.DNA unwinding point()D.S.DNA rewinding point()factor point,原核生物RNApol(Core)的结构与功能,Enzyme Movement,DNA coding strand(),Rewinding point(),Unwinding point(),RNA binding site,RNA/DNA hybrid(),DNA template strand,10-17bp,原核生物RNApol(Core)的结构与功能,Enzyme Movement,RNA binding site,RNA/DNA hybrid(),I,E,
