BACTERIAL TRANSCRIPTION COMPLEXES

描述(申请人提供)：这项提案集中在细菌RNA聚合酶(RNAP)的转录启动和延伸。转录起始和延伸涉及一系列步骤：(I)RNAP与启动子DNA结合，产生RNAP-启动子封闭复合体；(Ii)RNAP解开启动子DNA，产生RNAP-启动子开放复合体；(Iii)RNAP合成RNA的第一个~8-15个核苷酸作为RNAP-启动子起始转录复合体，在每个核苷酸加成循环中，RNAP保持静止在启动子DNA上，并拉入相邻DNA；以及(Iv)RNAP打破其与启动子DNA的相互作用，并将RNA的其余核苷酸合成为RNAP-DNA延伸复合体，在每个核苷酸加成循环中，RNAP在DNA上向前移动。这些步骤中的每一步都是转录调控者的潜在目标。理解转录起始、转录延伸和转录调控将需要定义蛋白质和DNA在每一步的结构转换，定义结构转换的动力学，并定义转录调控因子影响结构转换的机制。这项工作将利用系综和单分子荧光共振能量转移、单分子纳米操纵、生化方法和遗传学方法来解决五个特定目标：特定目标1：确定压缩特定目标的机制2：确定压缩特定目标的作用3：确定RNAP活性中心裂解负载特定目标的机制4：检测和分析RNAP活性中心构象循环特定目标5：确定转录调控因子ppGpp和Dks A的靶标和机制该结果将有助于理解细菌转录和转录调控，并将有助于设计和合成细菌转录小分子抑制物，应用于抗菌治疗。由于细菌RNAP亚基在序列、结构和机制上与真核RNAP亚基具有相似性，因此这一结果也将有助于理解真核转录和转录调控。公共卫生相关性：细菌RNA聚合酶(RNAP)是一种分子机器，执行细菌基因表达和细菌生长所必需的反应。一类重要的广谱抗菌治疗药物通过抑制RNAP发挥作用。这项工作将为理解RNAP的作用机制和合理设计通过抑制RNAP发挥作用的改进和新型广谱抗菌剂提供必要的信息。

DESCRIPTION (provided by applicant): This proposal focusses on transcription initiation and elongation by bacterial RNA polymerase (RNAP). Transcription initiation and elongation involve a series of steps: (i) RNAP binds to promoter DNA, yielding an RNAP-promoter closed complex; (ii) RNAP unwinds promoter DNA, yielding an RNAP-promoter open complex; (iii) RNAP synthesizes the first ~8 -15 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) RNAP breaks its interactions with promoter DNA and 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. Each of these steps is a potential target for transcriptional regulators. Understanding transcription initiation, transcription elongation, and transcriptional regulation will require defining the structural transitions in protein and DNA at each step, defining kinetics of structural transitions, and defining mechanisms by which transcriptional regulators affect structural transitions. The proposed work will use ensemble and single-molecule fluorescence resonance energy transfer, single-molecule nanomanipulation, biochemical methods, and genetic methods to address five specific aims: Specific Aim 1: Determination of the mechanism of scrunching Specific Aim 2: Determination of the role of scrunching Specific Aim 3: Determination of the mechanism of RNAP active-center-cleft loading Specific Aim 4: Detection and analysis of RNAP active-center conformational cycling Specific Aim 5: Determination of the target and mechanism of transcriptional regulators ppGpp and DksA The results will contribute to understanding bacterial transcription and transcriptional regulation, and will contribute to design and synthesis of small-molecule inhibitors of bacterial transcription, for application in antibacterial therapy. Since bacterial RNAP subunits show sequence, structural, and mechanistic similarities to eukaryotic RNAP subunits, the results also will contribute to understanding eukaryotic transcription and transcriptional regulation. PUBLIC HEALTH RELEVANCE: Bacterial RNA polymerase (RNAP) is a molecular machine that carries out reactions essential for bacterial gene expression and bacterial growth. An important class of broad-spectrum antibacterial therapeutic agents functions by inhibiting RNAP. The proposed work will provide information essential for understanding the mechanism of action of RNAP and for rational design of improved and novel broad-spectrum antibacterial agents that function by inhibiting RNAP.