Structure Function Studies of a RNA Antiterminator Bulge

描述(申请人提供)：最近，一个独特的监管要素在 对革兰氏阳性菌中tRNA合成酶的转录进行了鉴定。 未带电的tRNA与mRNA5‘之间发生一种新的RNA-RNA相互作用 许多革兰氏阳性tRNA合成酶的前导区。这种互动导致了 转录的反终止和基因的完全通读。如果没有 这种相互作用(即在只存在带电的tRNA的情况下)、转录 就会发生终止。它的序列和二级结构依赖性 反终止表明一种明确的、依赖于序列的相互作用。然而， 基于这些研究，它也似乎总体上是三维的 前导区的结构及其与不带电荷的tRNA的复合体发挥着作用 在抗终末期功能中的关键作用。假设是存在的 抗终止剂突起中一个重要的三级结构/功能相关性 引线区域的一部分。通过研究突变序列的结构 与野生型相比，抗堕胎能力下降，海因斯博士可以开始 构建一种结构/功能关系。具有进一步的结构性 信息，她可以观察与tRNA相互作用时的变化，并开始 对药物抑制剂进行分析并提出建议。这个项目的长期目标是 破坏tRNA/mRNA与小分子的相互作用和功能 已经针对该系统，使用在 这些研究。这样的研究将导致新型抗生素的开发。 具体目标一：海因斯博士将研究 抗终止子凸起突变体，突变与基因有关 发挥书房的功能作用。突变体的结构将是 与野生型相比，为了增加对隆起的认识 此系统的结构/功能关系。具体目标二：使用任何一种 完全修饰的tRNA或简化的tRNA受体茎模型RNA她将 研究tRNA/反终止子凸起相互作用的溶液行为。 她将使用天然凝胶、荧光和核磁共振来研究这种相互作用。 具体目标三：她将确定tRNAIBulge序列和结构 体外功能相互作用和体内抗肌腱的要求。 具体目标四：她将开始研究小RNA结合的影响 配体可能对反终止剂的溶液行为具有单独的或 与tRNA受体茎结合。

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.