分子生物学第四章1

基因的表达基因的表达 Gene expression第第4章章 RNA转录转录(RNA transcription)4.1 基本概念基本概念4.2 原核生物原核生物RNA转录启动子转录启动子 4.3 真核生物真核生物RNA转录启动子转录启动子4.4 Transcription Elongation 4.5 Transcription termination 4.6 Anti-termination in Rho-dependent terminator of Prokaryotes 4.7 Pre-RNA processing in Eukaryotes4.1 基本概念基本概念 基因表达的第一步基因表达的第一步 以以D.S.DNA中的一条单链作为转录的模板中的一条单链作为转录的模板 在依赖在依赖DNA的的RNA聚合酶的作用下聚合酶的作用下 模板单链模板单链 DNA的极性方向为的极性方向为3 5,而非模板单链而非模板单链 DNA的极性方向与的极性方向与RNA链相同，均为链相同，均为5 3.DNA(书写书写DNA序列时，仅写非模板序列，可不注明极性方向序列时，仅写非模板序列，可不注明极性方向)3-TACTCAT-5RNA 5-AUGAGUA-35-ATGAGTA-3 Non-template(sense strand)template(antisense strand)按按A U，C G 配对的原则，合成配对的原则，合成RNA分子分子 某一基因只以一条单链某一基因只以一条单链DNA 为模板进行转录（不对称转录）为模板进行转录（不对称转录）RNA的转录包括的转录包括promotion,elongation,termination 三步骤三步骤 从启动子（从启动子（promoter）到终止子（）到终止子（terminator）称为）称为 转录单位转录单位（transcriptional unit）原核生物中的转录单位多为原核生物中的转录单位多为 polycistron in operonpolycistron in operon 转录原点记为转录原点记为+1，其上游记为负值，下游记为正值，其上游记为负值，下游记为正值 真核生物中的转录单位多为真核生物中的转录单位多为monocistron,No operonmonocistron,No operon+1-10+10upstreamstart pointdownstreamDiscovery of messenger RNAT2 phage E.coli Host RNA stopped T2 RNA starting 1956.Elliot Volkin&Lawrence Astrachan1955 Brachet synthesis of protein depend on the RNA 1955 Goldstein&PlautRNA synthesized in nucleus and protein synthesized in cytoplasm Shift to N14C12 label RNA with P32 or protein with S35 T2 phage Brenner,Jacob&Meselson(1961)after incubation and break up E.coli add N14C12 ribosome as CkN15C13 Heavy ribosome in E.colidensity gradient resultsbottom topP32 T2 RNALight ribosomeHeavy ribosomeBrenner,Jacob&Meselson(1961)bottom topS 35 T2 proteinLight ribosomeHeavy ribosomeBrenner,Jacob&Meselson(1961)T2侵染E.coli后 核糖体上负载的RNA的碱基比与T2相似，而与E.coli相差甚远 E.coli 停止合成RNA&protein T2利用寄主细胞的转录元件和核糖体合成RNA&protein 核糖体上负载的RNA 是以T2DNA为模板转录的（其碱基组成反映了DNA的核苷酸序列）足以携带一个基因的遗传信息，指导T2蛋白质的合成 至少暂时连接在核糖体上 核糖体无特异性 +N14C12 ribosome as CKN15C13 N15C13+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 的结构与功能的结构与功能(Prok.E.coli)a)promoter 由两个重要部分组成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基因表达调控的正控制位点CAP:cAMP Acceptor Protein(环化环化AMP受体蛋白受体蛋白)Promoter region identified by DNAase method RNA polymerase (no NTP)DNA sequencing DNase digestion DNA+dissolve Promoter region including Sextama Box;RNApol.recognition site(R site)TTGAC(Sextama Box)-35 site RNApol.loosely binding sitePribnow Box;TATAAT(pribnow Box)-10 site RNApol.firmly binding site(B site)Initiation site;+1 RNA transcriptional start point(I site)A/G-35(R)-10(B)+1(I)RNA R -35 B-10I+1 Sextama BoxPribnow BoxInitiationb)CAP-cAMP binding site(Activator region,AR)ARI+CAP-cAMP AR I：CAP-cAMP 的强结合位点（的强结合位点（-70 -50）AR II：CAP-cAMP 的弱结合位点（的弱结合位点（-50 -40）cooperative effect提高提高ARII 的结合效率的结合效率IR-70 AR I-40AR II IR-50 AR II+CAP-cAMP 复合体复合体 促使促使Sextama Box 附近附近GC岛区的双螺旋结构稳定性降低，岛区的双螺旋结构稳定性降低，Pribnow Box 的解链温度降低，利于转录启动的解链温度降低，利于转录启动 ARI ARII GC Island Sextama Pribnow AR II+CAP-cAMP promotionRNApol.into Sextama Boxinto Pribnow Boxstarting transcription RNApol-CTDCAP-cAMPARI ARIIARI ARIIActivation transcriptionDuring 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.c)Pribnow Box 的突变与遗传效应的突变与遗传效应-T82T84G78A65C54A45-T80A95T45A80A50T96-c(A/G)t-(100)R B I-C17 TATAATG-pRE AAGTATCAC LRNA-str TAAAATCBio-p98 TAAATT C LRNA-tyr TATGAT C Cgal TATGGT T 14/16 down mutation-Clac TATGTTA ALRNA-trp TTAACTCLac-p115 TATTGTA-CiR TACACT C G C2/16 up mutation Pribnow Box 中中A/TT/A,改变了碱基堆积状况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（与模板链的结合效率，转录效率上升（上升上升突变突变）Pribnow Box 含有含有G/C,site II 必须有必须有 CAP-cAMP,以改变双螺旋体的稳定性以改变双螺旋体的稳定性 A/T G/C 双螺旋体稳定性双螺旋体稳定性 加强加强,转录率下降转录率下降(下降突变下降突变)l Sextama Box 与与Pribnow Box 间距间距17bp,有利于有利于RNApol启动启动l Sextama Box or Pribnow Box mut.间距趋近于间距趋近于17 bp，up mutation间距远离于间距远离于17 bp，down mutation17bp的间距的间距较较17bp的序列的序列对转录更为重要对转录更为重要Sextama Box and Pribnow Box mut.转录率下降转录率下降100X 转录率下降转录率下降1000X4.2.2 RNA polymerase in Prok.Core EnzymeHolo Enzyme for elongationfor initiation依靠静电作用力依靠静电作用力 依靠空间结构依靠空间结构非专一性与非专一性与DNA 结合结合专一性与专一性与 DNA结合结合结合常数；结合常数；1011/mol结合常数；结合常数；1014/mol半衰期；半衰期；60衰期；数小时衰期；数小时cover75bp 因子：因子：重复使用重复使用(Reusable)使使 Holo-enzyme识别识别Sextama Box,与模板链结合与模板链结合 修饰修饰 RNApol 构型，构型，降低全酶与降低全酶与DNA的非专一性结合力的非专一性结合力（107/mol）增强全酶与增强全酶与R,B site的专一性结合力的专一性结合力(1014/mol)导致导致RNA链的延伸缓慢链的延伸缓慢Sigma factor controls binding to DNAlSigma factor is the subunit of bacterial RNA polymerase needed for initiationlA 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：因子：促使促使RNApol与与DNA 模板链结合模板链结合 位于前端的位于前端的因子使双链解链为单链因子使双链解链为单链 位于尾端的位于尾端的因子使单链新聚合为双链因子使单链新聚合为双链 核心酶的组建因子核心酶的组建因子+2+Arg-COOH R-PP(ADP)-R-PP(40)ADP-核糖基化核糖基化 ADP核糖基聚合酶核糖基聚合酶 降低与降低与promoter的特异结合力的特异结合力因子：因子：促进促进RNA pol+NTP RNA elongation 完成完成 NMP之间的磷酸脂键的连接之间的磷酸脂键的连接 Editing 与与 Rho()因子竞争因子竞争RNA 3-end 构成构成Holo Enzyme后，后，因子含有因子含有 两个位点两个位点I site(Rif S);E site(Rif R);要求高浓度的要求高浓度的ATP or GTP专一性地结合专一性地结合ATP or GTP对对 NTP 非专一性结合非专一性结合Rifampin是是RNA合成起始的抑制剂合成起始的抑制剂 因子：因子：强碱性亚基强碱性亚基 促使促使RNA pol与非模板链（与非模板链（sense strand）强强结合结合 受受K酶抑制酶抑制 Holo Enzyme 含有五个功能位点含有五个功能位点 lsense strand DNA binding point()lDNA/RNA hybrid site（形成磷酸二酯（形成磷酸二酯 键）键）()lD.S.DNA unwinding point()lD.S.DNA rewinding point()lfactor point 原核生物原核生物RNApol(Core)的结构与功能的结构与功能Enzyme MovementDNA coding strand()Rewinding point()Unwinding point()RNA binding site RNA/DNA hybrid()DNA template strand 10-17bp 原核生物原核生物RNApol(Core)的结构与功能的结构与功能Enzyme MovementRNA binding site RNA/DNA hybrid()IEE site(Rif R);非专一性结合非专一性结合 NTPI site(Rif S);专一性地结合专一性地结合ATP/GTPPurification of the individual subunits of E.coli RNA polymerase in Urea electrophoresis buffer (Alfred heil&Ziling 1970)Separation and reconstitution of RNA polymerase to locate the determination of Rifampin resistance The reconstituted RNA polymerase was Rif S,regardless of the origin of the other subunits The subunit is obviously the determinant of Rifampin sensitivity or resistance Recombination the+from Rif R E.coli with from Rif R E.coli the+from Rif R E.coli with from Rif S E.coli