BACTERIAL TRANSCRIPTION COMPLEXES

描述（由申请人提供）：该提案侧重于细菌RNA聚合酶（RNAP）的转录起始和延伸。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-启动子封闭复合物;（ii）RNAP解开启动子DNA，产生RNAP-启动子开放复合物;（iii）RNAP使用“挤压”机制合成RNA的前8 - 15个核苷酸作为RNAP-启动子起始转录复合物，其中RNAP在启动子DNA上保持固定，并在每个核苷酸添加循环中拉入相邻的DNA;和（iv）RNAP破坏其与启动子DNA的相互作用，并使用“步进”机制将RNA的剩余核苷酸合成为RNAP-DNA延伸复合物，其中RNAP在每个核苷酸添加循环中在DNA上向前移动。这些步骤中的每一个都是转录调节因子的潜在靶点。理解转录起始、转录延伸和转录调控需要定义蛋白质和DNA在每一步的结构转变，定义结构转变的动力学，并定义转录调控因子影响结构转变的机制。拟议的工作将使用系综和单分子荧光共振能量转移，单分子纳米操纵，生物化学方法和遗传学方法来解决五个具体目标：具体目标1：确定挤压的机制具体目标2：确定挤压的作用具体目标3：确定RNAP活性中心裂缝加载的机制具体目标4：RNAP活性中心构象循环的检测与分析特异性目的5：确定转录调控因子ppGpp和DksA的作用靶点和作用机制研究结果将有助于理解细菌的转录和转录调控，并有助于设计和合成小分子细菌转录抑制剂，用于抗菌治疗。由于细菌RNAP亚基与真核RNAP亚基具有序列、结构和机制上的相似性，因此这些结果也将有助于理解真核生物的转录和转录调控。公共卫生相关性：细菌RNA聚合酶（RNAP）是一种分子机器，进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剂通过抑制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.