Protein/Protein Interactions in Prokaryotic Gene Control

描述（由申请人提供）：细菌中的基因表达关键取决于RNA聚合酶的σ亚基，其与核心酶结合形成全酶。 拟议的研究旨在研究功能上重要的相互作用的西格玛与核心，与转录调节因子，并与DNA。 将开发遗传工具，以促进在最近确定的核心和全酶结构的背景下进行这种分析。 Sigma通过与保守启动子元件的直接接触介导启动子识别和启动能力开放（熔化）复合物的形成。 为了执行这些功能，西格玛与核心酶，这是高度保守的细菌到man. One目标的建议的研究是使用最近开发的遗传工具，以进一步研究一个这样的接触：最近发现的主要西格玛因子（西格玛70）和β亚基的保守结构域之间的相互作用。RNAP需要这种相互作用来识别主要类别的σ 70依赖性启动子。 Sigma 70具有两个DNA结合结构域（保守区2和4），其与两个离散的启动子元件进行序列特异性接触。 拟议研究的另一个目标是进一步开发遗传分析，以促进这些蛋白质-DNA相互作用的解剖。 特别地，介导启动子解链的σ 70的区域2具有结合双链和单链DNA的能力。将制定办法，以确定这两项活动的关键决定因素。 最后，最近的工作表明，sigma 70的功能并不限于转录的起始阶段，但对sigma 70的这些额外作用知之甚少。 对噬菌体λ晚期启动子的研究揭示了σ 70在早期延伸复合物中的存在，并且该λ系统为研究σ 70在早期延伸中的功能提供了模型。拟议的研究解决了这项工作所提出的几个机械问题，以及调查在何种程度上这个lambda系统揭示了早期延伸复合体的一般特征。

DESCRIPTION (provided by applicant): Gene expression in bacteria depends critically on the sigma subunit of RNA polymerase, which combines with the core enzyme to form the holoenzyme. The proposed research is aimed at examining functionally important interactions of sigma with the core, with transcription regulators, and with DNA. Genetic tools will be developed to facilitate this analysis in the context of recently determined core and holoenzyme structures. Sigma mediates both promoter recognition and formation of the initiation-competent open (melted) complex through direct contact with conserved promoter elements. To carry out these functions sigma makes critical interactions with the core enzyme, which is highly conserved from bacteria to man. One goal of the proposed research is to use recently developed genetic tools to further examine one such contact: the recently uncovered interaction between the primary sigma factor (sigma 70) and a conserved domain of the beta subunit. This interaction is required for RNAP to recognize the major class of sigma 70-dependent promoters. Sigma 70 has two DNA-binding domains (conserved regions 2 and 4) that make sequence-specific contacts with two discrete promoter elements. Another goal of the proposed research is to further develop genetic assays to facilitate the dissection of these protein-DNA interactions. In particular, region 2 of sigma 70, which mediates promoter-melting, has the ability to bind both double- and single-stranded DNA. Approaches will be developed to identify critical determinants of these two activities. Finally, recent work has indicated that the functions of sigma 70 are not limited to the initiation phase of transcription, but relatively little is known about these additional roles of sigma 70. Studies of the bacteriophage lambda late promoter have revealed the presence of sigma 70 in an early elongation complex, and this lambda system has provided a model for studying the functions of sigma 70 in early elongation. The proposed research addresses several mechanistic questions raised by this work, as well as investigating the extent to which this lambda system reveals general features of the early elongation complex.