Bacterial Transcription Complexes

项目摘要 这项建议涉及细菌RNA聚合酶的转录起始、延长和终止。 (RNAP)。 在转录启动过程中，RNAP：(I)与启动子DNA结合，形成RNAP-启动子封闭复合体； 解开启动子DNA，产生RNAP-启动子开放复合体；(Iii)合成第一个~11个核苷酸 RNA作为RNAP-启动子的初始转录复合体，使用一种“压缩”机制，其中RNAP 在每个核苷酸加成循环中，在启动子DNA上保持静止，并拉入相邻的DNA； 从发起人那里逃脱。在转录延伸过程中，RNAP合成RNA的剩余核苷酸 作为RNAP-DNA伸长复合体，使用RNAP在DNA上向前移动的“步进”机制 在每个核苷酸加成循环中。在转录终止时，RNAP停止合成RNA，释放RNA， 从DNA中分离出来。这些反应中的每一个都是监管机构的目标。了解抄写 起始、延长、终止和调节需要确定蛋白质和蛋白质的结构转变 DNA在每个反应中的作用，结构转变的动力学，以及调节剂影响的机制 结构转型。 在当前阶段，我们定义了转录起始位置选择的结构基础--从头转录 起始、非规范起始核苷酸依赖的转录起始、起始转录和第II类 转录激活；我们开发了高通量测序方法，使 用于转录的DNA序列决定因素的分析；我们开发了多重交联法 能够对转录过程中的蛋白质-DNA相互作用进行全面分析；我们开发了系综和 能够监测RNAP钳和RNAP触发环的单分子荧光分析 并定义了小分子抑制剂的结合部位和作用机理。 抄写。 拟议的工作将以本期的调查结果为基础。拟议中的工作将使用x射线。 结晶学、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RNAP在延长、暂停和终止过程中的易位 具体目标4：分析RNAP钳在延长、暂停和终止过程中的构象 具体目标5：分析RNAP触发器环在延伸、暂停和终止过程中的构象

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