RNA-BASED MECHANISMS OF TRANSCRIPTION ELONGATION CONTROL

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。这项提案的目标是通过建立一个在联邦资金RO1水平上具有竞争力的研究计划，在肯塔基州建立研究能力。该项目旨在了解转录延伸控制的机制及其进化。我们建议对最初在噬菌体HK022中发现的一种独特的转录延伸控制机制进行表征。HK022是Lambda家族的一种温带噬菌体，可感染大肠杆菌。大多数Lambda噬菌体家族的成员使用噬菌体编码的蛋白通过抑制转录终止来促进基因的表达。HK022不需要抗终止蛋白，但依赖于新生RNA转录本中的位点与宿主RNA聚合酶的直接相互作用。这些位置称为PUT，分别位于两个早期操纵子中，由两个茎和环组成，由一个不成对的碱基隔开。我S的网站结构很重要，对它们的功能很重要。破坏碱基配对的突变减少了抗终止性，而重新建立碱基配对的次级突变则恢复了终结者的通读。防止抗终止的宿主突变体仅发生在位于RNA聚合酶β亚基氨基末端的高度保守的锌指结构域中。这些和其他结果表明，β主要锌指识别Put RNA，该结构域在转录终止中具有普遍作用。通过比较野生型大肠杆菌和携带锌指突变的细胞的基因表达谱，我们希望确定这个高度保守的区域的细胞靶点。为了增加我们对RNA介导的抗终止的理解和特定RNA结构元件的重要性，我们将识别和表征更多的抗终止子RNA的例子。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this proposal is to build research capacity in the state of Kentucky by establishing a research program that is competitive at the RO1 level of federal funding. The project is designed to understand mechanisms of transcription elongation control and their evolution. We propose to characterize a unique mechanism of transcription elongation control originally discovered in the bacteriophage HK022. HK022 is a temperate phage of the lambda family that infects Escherichia coli. Most members of the lambda phage family use phage encoded proteins to promote gene expression by suppressing transcription termination. HK022 has dispensed with the requirement of an antitermination protein but relies on the direct interaction of sites in the nascent RNA transcript with the host RNA polymerase. These sites, called put, are located in each of the two early operons and are comprised of two stem and loops separated by an unpaired base. The structure of the sites i s important for their function. Mutations that disrupt base pairing reduce antitermination and secondary mutations that reestablish base pairing restore terminator read through. Host mutants that prevent antitermination occur exclusively in a highly conserved zinc-finger domain located at the amino terminus of the beta prime subunit of RNA polymerase. These and other results suggest that the beta prime zinc finger recognizes put RNA and that this domain has a general role in transcription termination. By comparing the gene expression profiles of wild type E. coli with cells that carry mutations in the zinc finger, we hope to identify cellular targets of this highly conserved domain. To increase our understanding of RNA-mediated antitermination and the importance of specific RNA structural elements, we will identify and characterize additional examples of antiterminator RNAs.