Nucleic Acid Structure-Function in Gene Expression

描述（由申请人提供）：我们研究的长期目标是 了解大肠杆菌的转录后调控。的 总体目标是解决当前和根本问题， 翻译和mRNA加工/降解。我们把注意力集中在一个 编码来自丝状噬菌体F1基因组的六个基因的转录单位， 大量转录，但仅在转录后进行调节 以产生不同数量的蛋白质。适当规范这些 需要基因来平衡噬菌体基因表达并允许噬菌体 在宿主体内保持持续感染。首先，拟议的研究将 在6个基因中的最后2个中完成对照的研究。这些基因是 编码必需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 包括mRNA的稳定性。我们将进一步证明RNA 结构明显抑制了较小基因的翻译和识别 多种因素限制了编码这两种基因的mRNA的内部起始， 基因.更广泛的兴趣，这将阐明如何调控基因表达 在读码框内重叠的基因对中，大量存在于细菌、噬菌体和质粒中 基因组，已经实现。其次，拟议的研究将重点放在 这些非常丰富的噬菌体mRNA进行mRNA降解的途径。的 具体的原理是，这些mRNA将反映对细胞的主要活性， 感染宿主的衰变机制，更重要的是， 说明衰变的5'到3'波是衰变的主要途径 细菌的mRNA。信使RNA衰变已被证明是一个重要参数， 决定基因表达水平，并允许对细胞的快速反应。 信号信号，但直到最近加速进展，一直是最慢的 主要的基因调控过程。目前 该领域有望取得重大进展。作为这项工作的一部分，我们将定义 f1 mRNA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会找出梅杰 在核酸内切和核酸外切衰变中起作用的酶， 规划体外重建研究，确定衰变途径， 较小的f1 mRNA底物。在一个新的方向，我们将跟踪f1 mRNA衰变 和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.