Nucleic Acid Structure-Function in Gene Expression

描述(申请人提供)：我们研究的长期目标是 了解了大肠杆菌的转录后调控。这个 总的目标是解决当前和根本的问题 翻译和信使核糖核酸的加工/衰退。我们将注意力集中在一个 编码丝状噬菌体F1基因组中六个基因的转录单位 转录数量巨大，但转录后仅受调控 以产生不同数量的蛋白质。对这些进行适当的监管 需要基因来平衡噬菌体基因的表达，并允许噬菌体 在其宿主中保持持续感染。首先，拟议的研究将 完成对6个基因中最后2个基因的对照研究。这些基因是一种 编码基本DNA复制基因的框内重叠对 相反的功能。目前的证据表明，这些基因在 几点，包括信使核糖核酸的稳定性。我们将强化证据，证明RNA 结构抑制小基因的翻译，并明显识别 多种因素限制了编码这两种基因的信使核糖核酸的内部启动 基因。更有意义的是，这将阐明基因表达的调控 在框内重叠的基因对中，在细菌、噬菌体和质粒中大量存在 基因组，是实现的。第二，拟议的研究将主要集中在 这些非常丰富的噬菌体mRNAs经历mRNA降解的途径。这个 具体的理由是，这些mRNA将反映出 受感染宿主的腐烂机制，更重要的是，噬菌体mRNAs 说明一个5‘到3’的衰变波，它是衰变的主要途径 细菌的核糖核酸。信使RNA衰变已被证明是一个重要的参数 确定基因表达水平并允许对细胞的快速反应 发出信号，但直到最近进展加快，一直是最慢的 有待研究的主要基因调控过程。目前， 菲尔德准备取得重大进展。作为这一努力的一部分，我们将定义 F1mRNA衰变途径的步骤，主要目的是测试提出的 衰变的分子模型。我们将辨认梅杰的乳沟 在核内和核外溶解腐烂中起作用的酶，以及作为 规划体外重建研究，定义衰变途径 较小的F1 mRNA底物。在一个新的方向上，我们将跟踪F1 mRNA的衰变 以及细菌细胞内的降解体定位。

DESCRIPTION (Provided by applicant): The long term goal of our research has been to understand post-transcriptional regulation in Escherichia coli. The general objective is to address current and fundamental questions in translation and mRNA processing/decay. We have focused attention on a transcription unit encoding six genes from the filamentous phage f1 genome that are transcribed in huge amounts but regulated exclusively after transcription to yield proteins made in different amounts. Appropriate regulation of these genes is required to balance phage gene expression and permit the phage to maintain a persistent infection in its host. First, the proposed research will complete studies of controls in the last 2 of the 6 genes. These genes are an in-frame overlapping pair encoding essential DNA replication genes with opposing functions. Present evidence indicates that the genes are regulated at several points, including mRNA stability. We will strengthen evidence that RNA structure inhibits translation of the smaller gene and identify apparently multiple factors that limit internal initiation on the mRNA encoding both genes. Of broader interest, this will clarify how regulation of gene expression in in- frame overlapping gene pairs, numerous in bacterial, phage and plasmid genomes, is achieved. Second, the proposed research will focus heavily on the pathway by which these very abundant phage mRNAs undergo mRNA degradation. The specific rationales are that these mRNAs will reflect a major activity on the decay machinery in infected hosts, and more important, the phage mRNAs illustrate a 5' to 3' wave of decay that is the predominant pathway for decay of bacterial mRNAs. Messenger RNA decay has proven an important parameter that determines levels of gene expression and permits rapid responses to cellular signalling signals, but until recent accelerated progress, has been the slowest of the principal gene regulatory processes to be worked out. Currently, the field is poised to make major progress. As part of this effort, we will define the steps of the f1 mRNA decay pathway, the major aim being to test proposed molecular models for decay. We will identify the cleavages made by major enzymes with functions in endo- and exonucleolytic decay, and as part of planning for in vitro reconstitution studies, define the decay pathways for smaller f1 mRNA substrates. In a new direction, we will follow f1 mRNA decay and degradosome localization within the bacterial cell.