Structure Function Studies of a RNA Antiterminator Bulge

描述（由申请人提供）：最近， 鉴定了革兰氏阳性菌中tRNA合成酶的转录。 一种新的RNA-RNA相互作用发生在不带电荷的tRNA和mRNA 5'端之间。 许多革兰氏阳性tRNA合成酶前导区。这种相互作用导致 转录的抗终止和基因的完全通读。没有 这种相互作用（即在仅存在带电荷的tRNA的情况下）、转录 终止发生。这个序列和二级结构依赖性 抗终止作用表示确定的序列依赖性相互作用。然而，在这方面， 根据这些研究，似乎整体的三维 前导区的结构及其与不带电荷的tRNA的复合物起着重要的作用。 在抗终止功能中的关键作用。假设是， 在抗终止子凸起中的关键三级结构/功能相关性 领导者区域的一部分。通过研究突变序列的结构， 与野生型相比，抗终止能力降低，海因斯博士可以开始 构建结构/功能关系。进一步结构化 信息，她可以看到与tRNA相互作用后的变化，并开始 分析并提出药物抑制剂。该项目的长期目标是 破坏tRNA/mRNA的相互作用和小分子的功能， 已经针对这个系统，使用获得的结构信息， 这些研究。这些研究将导致新抗生素的开发。 具体目标I：Hines博士将研究以下溶液结构： 抗终止子凸起突变体，其中突变与遗传学有关， 研究发挥功能作用。变种人的结构将是 与野生型凸起相比， 结构/功能关系。具体目标二：使用 完全修饰的tRNA或简化的tRNA受体茎模型RNA，她将 研究tRNA/antiterminator bulge相互作用的溶液行为。 她将使用天然凝胶，荧光和NMR研究这种相互作用。 具体目标III：她将确定tRNAIbulge序列和结构 体外功能性相互作用和体内抗末端化的要求。 具体目标IV：她将开始研究小RNA结合 配体可以具有单独的抗终止剂的溶液行为， 与tRNA受体茎复合。

DESCRIPTION (provided by applicant): Recently, a unique regulatory element in the transcription of tRNA synthetases in Gram-positive bacteria was identified. A novel RNA-RNA interaction occurs between uncharged tRNA and the mRNA 5' leader region of many Gram-positive tRNA synthetases. This interaction leads to antitermination of transcription and complete read-through of the gene. Without this interaction (i.e. in the presence of only charged tRNA), transcription termination occurs. The sequence and secondary structure dependence of this antitermination indicates a definite, sequence dependent interaction. However, based on these studies, it also appears as though the overall three-dimensional structure of the leader region and its complex with the uncharged tRNA plays a critical role in the antitermination function. The hypothesis is that there is a crucial tertiary structure/function correlation in the antiterminator bulge portion of the leader region. By studying structures of mutant sequences with decreased antitermination ability compared to the wild type, Dr.Hines can begin to construct a structure/function relationship. With further structural information, she can look at changes upon interaction with tRNA and begin to assay for and propose drug inhibitors. The long-range goal of this project is to disrupt the tRNA/mRNA interaction and function with small molecules that have been targeted to this system, using the structural information obtained in these studies. Such studies will lead to the development of novel antibiotics. Specific Aim I: Dr. Hines will investigate the solution structure of antiterminator bulge mutants where the mutation has been implicated by genetic studies to play a functional role. The structure of the mutants will be compared to the wild-type bulge in order to add to the knowledge of structure/function relationships for this system. Specific Aim II: Using either fully modified tRNA or a simplified tRNA acceptor stem model RNA she will investigate the solution behavior of the tRNA/antiterminator bulge interaction. She will investigate this interaction using native gels, fluorescence and NMR. Specific Aim III: She will determine tRNAIbulge sequence and structural requirements for functional interactions in vitro and antitennination in vivo. Specific Aim IV: She will begin to look at the effects small RNA binding ligands may have on the solution behavior of the antiterminator alone or complexed with tRNA acceptor stem.