Mechnanism of transcript elongation control by RfaH

描述(申请人提供)：许多基因的表达受到RNA聚合酶完成多达一百万个核苷酸聚合的能力的限制，使延伸出现在起始之后，成为基因表达的主要调节步骤。已经描述了几种使RNA聚合酶克服这一限制并变得“精通伸长”的辅助蛋白质因素。这一提议的重点是细菌蛋白RfaH，它是几个长毒力和生育操纵子的调节因子，在这些操纵子中，它优先增加远端基因的表达。我们已经证明RfaH与其招募序列OPs结合，暴露在伸长过程中暂停在OPs位点的RNA聚合酶的表面。在其募集之后，RfaH通过提高延伸率和抑制停顿来刺激OPs位点下游的转录。然而，RfaH仅适度地抑制终止。RfaH作用的详细机制被描述为“抗终止”，但它不同于其他抗终止因子，如lambdaN和lambdaQ，后者对延伸和终止都有深刻的影响，这一事实仍然不清楚。RfaH的招募模式和对伸长的影响都是独一无二的，因此深入了解RfaH的机制将有助于全面理解细菌和真核生物中转录本伸长的调控，其中RfaH同源基因参与伸长控制并定位于活跃的转录位点。在这项提案中，我们将使用生化、遗传和生物物理方法的组合来解决RfaH作用的几个方面。该项目的第一个目标是阐明RfaH影响其招募部位下游数千个核苷酸延伸的分子机制。需要回答的中心机制问题是RfaH是否与延长的RNA聚合酶一起旅行，或者它是否导致RNA聚合酶的构象变化，在RfaH从复合体解离后，RNA聚合酶持续数千个核苷酸加成步骤。该项目的第二个目标是通过找出RfaH是否在所有位置都以相似的方式影响转录或针对一组特定的调控信号来确定这种机制的普适性。该项目的第三个目标是绘制RfaH和转录延伸复合体之间的相互作用图，从而将RfaH机制置于其结构背景中。RfaH控制分泌分子、细胞壁成分、抗生素、毒力因子和蛋白质的表达，这些都是动员可传播质粒所必需的。因此，拟议的研究将对细菌生物学和进化的几个领域的研究产生积极影响，如细胞质外成分的合成、细菌毒力、横向基因转移和病原体的出现。

DESCRIPTION (provided by applicant): Expression of many genes is limited by the ability of RNA polymerase to complete polymerization of up to a million nucleotides, making elongation to emerge next to initiation as a major regulatory step in gene expression. Several accessory protein factors that allow RNA polymerase to overcome this limitation and become "elongation-proficient have been described. The focus of this proposal, the bacterial protein RfaH, is a regulator of several long virulence and fertility operons, where it preferentially increases the expression of distal genes. We have demonstrated that RfaH binds to its recruitment sequence, ops, exposed on the surface of the RNA polymerase paused at an ops site during elongation. Following its recruitment, RfaH stimulates transcription downstream of an ops site by enhancing elongation rate and suppressing pausing. However, RfaH only modestly inhibits termination. The detailed mechanism of RfaH action, described as "antitermination', remains obscure except for the fact that it is different from those of other antiterminators such as lambdaN and lambdaQ, which have profound effects on both elongation and termination. Both the recruitment mode and the effect of RfaH on elongation are unique, thus insights into the RfaH mechanism will contribute to the general understanding of the regulation of transcript elongation in bacteria and also in eukaryotes, where RfaH homologs are implicated in elongation control and localize to the actively transcribed sites. In this proposal, we will use a combination of biochemical, genetic, and biophysical approaches to address several aspects of RfaH action. The first goal of this project is to elucidate the molecular mechanism by which RfaH affects elongation thousands of nucleotides downstream of its recruitment site. The central mechanistic question to be answered is whether RfaH travels with the elongating RNA polymerase or if it causes a conformational change in the RNA polymerase that persists for thousands of nucleotide addition steps after RfaH dissociates from the complex. The second goal of this project is to determine how universal is this mechanism by finding out whether RfaH affects transcription similarly at all sites or is targeted to a particular set of regulatory signals. The third goal of this project is to map interactions between RfaH and the transcription elongation complex, thus placing RfaH mechanism in its structural context. RfaH controls the expression of the secreted molecules, components of the cell wall, antibiotics, virulence factors, and proteins required for the mobilization of transmissible plasmids. Proposed studies will therefore positively impact research in several areas of bacterial biology and evolution, such as synthesis of extracytoplasmic components, bacterial virulence, lateral gene transfer, and emergence of pathogens.