Mechnanism of transcript elongation control by RfaH

描述（由申请人提供）：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，使得延伸出现在起始旁边，作为基因表达的主要调控步骤。一些辅助蛋白因子允许RNA聚合酶克服这一限制，成为“伸长精通”已经被描述。该提案的重点是细菌蛋白RfaH，它是几个长毒力和生育操纵子的调节剂，在那里它优先增加远端基因的表达。我们已经证明，RfaH结合其募集序列，ops，暴露在表面的RNA聚合酶在延伸期间暂停在ops位点。在募集后，RfaH通过提高延伸率和抑制暂停来刺激ops位点下游的转录。然而，RfaH仅适度抑制终止。RfaH作用的详细机制，被描述为“反终止”，仍然不清楚，除了它不同于其他反终止剂，如lambdaN和lambdaQ，它们对延伸和终止都有深远的影响。RfaH的招募模式和对伸长的影响都是独特的，因此对RfaH机制的深入了解将有助于对细菌和真核生物转录物伸长调控的总体理解，其中RfaH同源物与伸长控制有关，并定位于活性转录位点。在本提案中，我们将使用生物化学，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影响其招募位点下游数千个核苷酸延伸的分子机制。要回答的核心机制问题是，RfaH是否与拉长的RNA聚合酶一起移动，或者它是否引起RNA聚合酶的构象变化，这种变化在RfaH与复合物分离后持续数千个核苷酸添加步骤。该项目的第二个目标是通过发现RfaH是否在所有位点上相似地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遍性。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，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、毒力因子和动员可传播质粒所需的蛋白质的表达。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，例如胞质外组分的合成、细菌毒力、侧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.