Bacterial Transcription Complexes

项目摘要 该提案涉及细菌RNA聚合酶的转录起始、延伸和终止 （RNAP）。 在转录起始中，RNAP：（i）与启动子DNA结合，产生RNAP-启动子封闭复合物;（ii） 解开启动子DNA，产生RNAP-启动子开放复合物;（iii）合成启动子DNA的前11个核苷酸。 RNA作为RNAP启动子初始转录复合物，使用“挤压”机制，其中RNAP 在启动子DNA上保持静止，并在每个核苷酸添加循环中拉入相邻的DNA;以及（iv） 从启动子中逃脱。在转录延伸中，RNAP合成RNA的剩余核苷酸 作为RNAP-DNA延伸复合物，使用RNAP在DNA上向前移动的“步进”机制， 在每个核苷酸添加循环中。在转录终止时，RNAP停止合成RNA，释放RNA， 并与DNA分离。这些反应中的每一个都是监管机构的目标。理解转录 起始、延伸、终止和调节将需要定义蛋白质中的结构转变， 每个反应中的DNA，结构转换的动力学，以及调节剂影响的机制 结构转型。 在本阶段，我们定义了转录起始位点选择的结构基础， 起始、非典型起始核苷酸依赖性转录起始、初始转录和II类 转录激活;我们开发了高通量测序方法， 分析转录的DNA序列决定子;我们开发了多重交联方法 这使得全面分析蛋白质-DNA相互作用的转录;我们开发了合奏， 能够监测RNAP钳和RNAP标记环的单分子荧光测定 在溶液中的构象;我们定义了小分子抑制剂的结合位点和机制， 转录。 拟议的工作将以本期的调查结果为基础。拟议的工作将使用x射线 晶体学，单分子生物物理学，生物化学和遗传学，以解决五个具体目标： 具体目标1：确定RNAP滑移的结构基础 具体目标2：确定RNAP易位的结构基础 具体目标3：分析延伸、暂停和终止中的RNAP易位 具体目标4：在延伸、暂停和终止中分析RNAP夹构象 具体目标5：在延伸、暂停和终止中分析RNAP双链-环构象

Project Summary This proposal addresses transcription initiation, elongation, and termination by bacterial RNA polymerase (RNAP). In transcription initiation, RNAP: (i) binds to promoter DNA, yielding an RNAP-promoter closed complex; (ii) unwinds promoter DNA, yielding an RNAP-promoter open complex; (iii) synthesizes the first ~11 nucleotides of RNA as an RNAP-promoter initial transcribing complex, using a "scrunching" mechanism in which RNAP remains stationary on promoter DNA and pulls in adjacent DNA in each nucleotide-addition cycle; and (iv) escapes from the promoter. In transcription elongation, RNAP synthesizes the remaining nucleotides of RNA as an RNAP-DNA elongation complex, using a "stepping" mechanism in which RNAP moves forward on DNA in each nucleotide-addition cycle. In transcription termination, RNAP stops synthesizing RNA, releases RNA, and dissociates from DNA. Each of these reactions is a target for regulators. Understanding transcription initiation, elongation, termination, and regulation will require defining the structural transitions in protein and DNA in each reaction, the kinetics of structural transitions, and the mechanisms by which regulators affect structural transitions. In the current period, we defined the structural basis of transcription start-site selection, de novo transcription initiation, non-canonical-initiating-nucleotide-dependent transcription initiation, initial transcription, and Class II transcription activation; we developed high-throughput-sequencing approaches that enable comprehensive analysis of DNA-sequence determinants for transcription; we developed multiplexed crosslinking approaches that enable comprehensive analysis of protein-DNA interactions in transcription; we developed ensemble and single-molecule fluorescence assays that enable monitoring of RNAP clamp and RNAP trigger-loop conformation in solution; and we defined binding sites and mechanisms for small-molecule inhibitors of transcription. The proposed work will build on the findings of the current period. The proposed work will use x-ray crystallography, single-molecule biophysics, biochemistry, and genetics to address five specific aims: Specific Aim 1: Determination of the structural basis of RNAP slippage Specific Aim 2: Determination of the structural basis of RNAP translocation Specific Aim 3: Analysis of RNAP translocation in elongation, pausing, and termination Specific Aim 4: Analysis of RNAP clamp conformation in elongation, pausing, and termination Specific Aim 5: Analysis of RNAP trigger-loop conformation in elongation, pausing, and termination