Identification and Characterization of Antiterminator RNAs

描述(由申请者提供)：这项建议实现了以下领域的计划目标：1)支持有价值的研究；2)让本科生接触研究；3)加强非研究密集型大学的研究环境。这个项目的目标是了解最初在噬菌体HK022中发现的一种独特的转录延伸控制机制。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早期基因的表达。HK022不编码抗终止蛋白，而是依赖于新生转录本中的位点与RNA聚合酶的直接相互作用。具体的假设是，基于RNA的抗终止子的活性取决于RNA聚合酶对新生转录本中序列和结构信息的识别。这一假说是基于以前的研究，1)已经表明破坏RNA中碱基配对的突变减少了抗终止，而重新建立碱基配对恢复终止子的次级突变2)抗终止RNA的交换片段极大地影响了活性，以及3)RNA介导的抗终止被大肠杆菌RNA聚合酶的β主亚单位的突变所阻断。这项建议的目的是确定基于RNA的反终止子所需的序列和结构元件。其具体目标是：1)确定更多的Lambdoid噬菌体中抗终止子RNA的例子；2)使用体内、体外和电子计算机方法来确定新发现的抗终止子RNA的结构和功能上的重要特征；以及3)完成两个新的具有基于RNA的抗终止子的噬菌体基因组的注释。在HK022中发现的抗终止子序列提供了通过直接修改转录装置来控制基因表达的RNA的独特例子。不寻常的基因调控模式是药物设计的潜在目标。因此，更好地了解抗终止子RNA及其被RNA聚合酶识别可能有助于发现或开发能够改变毒力基因表达从而减轻疾病进程的治疗剂。西肯塔基大学立志成为肯塔基州最好的综合性公立学校，在全国名列前茅。该项目支持这一使命，并将通过为本科生提供大量学习生物医学研究基础知识的机会，同时探索一种独特的基因表达控制机制，来改善西部大学的研究环境。在噬菌体HK022中发现的抗终止子序列提供了通过直接修改转录装置来控制基因表达的RNA的独特例子。不寻常的基因调控模式是药物设计的潜在目标。因此，更好地了解抗终止子RNA及其被RNA聚合酶识别可能有助于发现或开发能够改变毒力基因表达从而减轻疾病进程的治疗剂。

DESCRIPTION (provided by applicant): This proposal accomplishes the AREA program objectives of: 1) supporting meritorious research; 2) exposing undergraduates to research; and 3) strengthening the research environment in non-research intensive universities. The goal of this project is to understand a unique mechanism of transcription elongation control originally discovered in the bacteriophage HK022. Most members of the lambda phage family use phage-encoded proteins to promote early gene expression by suppressing transcription termination. HK022 does not encode an antitermination protein but relies instead on the direct interaction of sites in the nascent transcript with RNA polymerase. The specific hypothesis is that the activity of RNA-based antiterminators depends upon the recognition of sequence and structural information in the nascent transcript by RNA polymerase. This hypothesis is based upon previous studies that 1) have shown that mutations that disrupt base pairing in the RNA reduce antitermination and secondary mutations that re-establish base pairing restore terminator read through 2) interchanging segments of antiterminator RNAs drastically affects activity, and 3) RNA-mediated antitermination is blocked by mutations in the beta prime subunit of E. coli RNA polymerase. The goal of this proposal is to identify the required sequence and structural elements of RNA based antiterminators. The specific aims are: 1) to identify additional examples of antiterminator RNAs in lambdoid phages; 2) to use in vivo, in vitro and in silico approaches to determine the structurally and functionally important features of the newly identified antiterminator RNAs; and 3) to complete the annotation of two new phage genomes that possess RNA-based antiterminators. The antiterminator sequences discovered in HK022 provide unique examples of RNAs that control gene expression by directly modifying the transcription apparatus. Unusual modes of gene regulation are potential targets for drug design. Therefore, a better understanding of antiterminator RNAs and their recognition by RNA polymerase may facilitate the discovery or development of therapeutic agents capable of altering the expression of virulence genes and thus attenuating disease processes. Western Kentucky University aspires to be the best comprehensive public institution in Kentucky and among the best in the nation. This project supports this mission and will enhance the research environment at Western by providing undergraduate students with numerous opportunities to learn the fundamentals of biomedical research while exploring a unique mechanism of gene expression control. The antiterminator sequences discovered in phage HK022 provide unique examples of RNAs that control gene expression by directly modifying the transcription apparatus. Unusual modes of gene regulation are potential targets for drug design. Therefore, a better understanding of antiterminator RNAs and their recognition by RNA polymerase may facilitate the discovery or development of therapeutic agents capable of altering the expression of virulence genes and thus attenuating disease processes.