分子生物学第四章transcriptionelongationppt课件

l 非进行型复合体（Non-processive complex）,未被激活的基因的表达方式,RNApol II pausing at 5-end of gene，并表现为DRBR,- 是对基因非激活转录的中止机制,- 维持正常生理代谢进行的机制,- 与转录启动复合体的非完整性有关（abortive TIC）,Terminator: A sequence of DNA, presented at the end of the transcript, that causes RNA polymerase to terminate transcription.,4.4 Transcription termination,4.4.1 Termination in prokaryotes,Palindromic sequence,G/C rich region that forms hairpins of varying lengths (7-20bp) in RNA,G/C rich hairpin are followed by a run of U residues in RNA,Structure,Rho-independent terminator (Intrinsic terminator),Function,G/C rich transcription delay,RNA stem-loop & polyA/U RNApol leave, inhibited by K factor RNApol & DNA separated,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,Rho-factor (),l Hexamer 55kd neutral,l Contest RNA 3-end with factor,l ATPase-like, GTPase-like activity needs spacer of 50 Nt,l 转录终止效率取决于紧随回文的下游序列,-保证 RNA / DNA 具有一定松弛的结合力,-过低的结合力 成为 Rho-independent terminator,-过高的结合力 引起 RNApol read-through,-各依赖 Rho 因子的终止子间的效率差异取决于 终止子区DNA / RNA 间结合力的差异,Model of rho-dependent termination,b) When the hairpin forms in the transcript, the polymerase pauses, giving Rho a chance to catch up.,c) Rho helicase unwinds the RNA-DNA hybrid and releases the transcript.,a) Rho binds to the transcript and pursues the polymerase.,The action of rho factor may create a link between transcription and translation when a rho-dependent terminator lies soon after a nonsense mutation.,Antitermination depends on specific sites,4.4.2 Anti-termination in Rho-dependent terminator of Prokaryotes,Antitermination proteins: allow RNA polymerase to transcribe through certain terminator sites.,Antitermination can be used to control transcription by determining whether RNA polymerase terminates or reads through a particular terminator into the following region.,Switches in transcriptional specificity can be controlled at initiation or termination.,Ancillary factors bind to RNA polymerase as it passes certain sites. The nut site consists of two sequences. NusB-S10 join core enzyme as it passes boxA. Then NusA and pN protein bind as polymerase passes boxB. The presence of pN allows the enzyme to read through the terminator, producing a joint mRNA that contains immediate early sequences joined to delayed early sequences.,RNA polymerase may alternate between initiation-competent and termination-competent forms as sigma and Nus factors alternatively replace one another on the core enzyme.,nusA与核心酶结合，但并不与全酶发生作用。当Sigma因子加入到酶复合体中，它代替了nusA蛋白质，重新组成了全酶。这也就是说RNA酶以准备起始和准备终止的交替形式存在，经历了如图所示的循环。当全酶与启动子结合后，释放了Sigma因子，产生了核心酶来合成RNA，当一个nusA蛋白质辩识了核心酶并与之结合，就产生了酶复合物。当 聚合酶与DNA结合时，nus成分不能被替换。当发生终止时，酶被释放，nus因子或者被释放，或者被sigma因子所取代。,phage regulatory region contains a cluster of trans & cis-factor,Np,Np,Cis-F：PR,OR, NuTR, TR, PRE, PRM, PLOL, NuTL, TL1, TL2, Pint Trans-F：CI, Cro, CII, N, Q, CIII, int, Xis,4.4.3 Principle of anti-termination inphage,N-p 结合在 NuT R1 (NuTL1) 位点,等待 RNApol,Np使RNApol变构并不能与Rho因子结合,RNApol 经过 terminator 并发生通读现象 （read-through ）,Host RNA polymerase transcribes lambda genes and terminates at t sites. pN allows it to read through terminators in the L and R1 units; pQ allows it to read through the R terminator. The sites at which pN acts (nut) and at which pQ acts (qut) are located at different relative positions in the transcription units.,pQ通过在qut位点起作用。qut序列位于后期转录单位的起点。它的前部分位于起动子之中，后部分在被转录部分的开始。这暗示了pQ起作用与DNA的识别是联系着的，它作用的机理，至少涉及最初结合复合体，一定与pN不同。 pQ的基本作用是干扰暂停，一旦pQ对RNA聚合酶起作用，酶在包括rho-依赖性和真实终止子在内的所有位点处的暂停将缩短。这样，pQ没有直接作用于终止子本身，而是让酶迅速通过终止子，使核心酶和（或）辅助因子没有机会引起终止。,抗终止机制是否多发生于原核生物内？,4.5 Pre-RNA processing in Eukaryotes,4.5.1 概念,pre-RNA,capping tailing splicing methylation editing,生物学意义,mature RNA, prevent pre-RNA from digested by RNase,as template (protein translation),l interrupted gene,(interrupted RNA),remove introns (stop codon),RNA splicing is the process of excising the sequences in RNA that correspond to introns, so that the sequences corresponding to exons are connected into a continuous mRNA. pre-mRNA heterogeneous nuclear RNA (hnRNA) (核内不均一RNA) hnRNP (ribonucleoproteosome),Types & struc