CONTROL OF TRANSCRIPTION ANTITERMINATION IN E COLI

大肠杆菌中转录抗终止的控制

• 批准号: 3308339
• 负责人: ANDREW WRIGHT
• 金额: $ 16.6万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3308339
• 关键词: Bacillus subtilis; DNA footprinting; Escherichia coli; RNA binding protein; affinity chromatography; bacterial genetics; bacterial proteins; chimeric proteins; dimer; gene expression; nucleic acid structure; point mutation; polymerase chain reaction; protein structure; tissue /cell culture; transcription termination; western blottings

The bg1G gene product is an RNA binding protein which functions as a transcriptional antiterminator in E. coli. We have identified the RNA target for this protein and now wish to study precisely how the protein interacts with the RNA. Preliminary indications are that the protein recognizes a region of RNA secondary structure with a bulged are being important. Other important regulators such as HIV Tat appear to also recognize such structures, however Bg1G protein does not seem to have a Tat-like RNA binding motif thus it may represent a novel family of RNA binding proteins. To study how Bg1G interacts with RNA we will purify the protein using a gene fusion approach since it will allow affinity purification. The purified protein will be used to study interaction with RNA by gel retardation and chemical footprinting. In parallel we will also purify a B.subtilis protein, SacY, which has properties similar to Bg1G and which shares significant homology with it. We will carry out a comparative series of binding studies to try to determine the elements within the proteins that are important for binding specificity. The SacY RNA target will be used in these studies as well since it differs in several respects from the Bg1G target. We have found that a specific mutation (C to A change) at position 50 in the Bg1G target RNA eliminates Bg1G recognition without altering the overall RNA structure. We intend to look for mutations in Bg1G that suppress the RNA alteration and thus hope to identify regions of contact. We will also search for mutants of Bg1G that have a dominant negative phenotype. These would provide additional support for the observation that Bg1G binds to RNA as a dimer. Finally we will look for mutations that alleviate any dominant negative effect with the aim of identifying sites important for dimerization or that interact with one another in the dimer.

bg1G 基因产物是一种 RNA 结合蛋白，其功能为 大肠杆菌中的转录抗终止子。 我们已经鉴定出RNA 该蛋白质的目标，现在希望精确研究该蛋白质如何 与 RNA 相互作用。 初步迹象表明该蛋白质 识别RNA二级结构的一个区域，其中有一个凸出的区域 重要的。 其他重要的调节因子，例如 HIV Tat 似乎也 识别此类结构，但是 Bg1G 蛋白似乎没有 Tat 样 RNA 结合基序因此可能代表一个新的 RNA 家族 结合蛋白。 为了研究 Bg1G 如何与 RNA 相互作用，我们将纯化 使用基因融合方法的蛋白质，因为它将允许亲和力 纯化。 纯化的蛋白质将用于研究相互作用 通过凝胶阻滞和化学足迹法与 RNA 结合。 与此同时，我们 还将纯化枯草芽孢杆菌蛋白 SacY，其具有类似的特性 与 Bg1G 具有显着的同源性。 我们将进行 尝试确定元素的一系列比较性结合研究 在对结合特异性很重要的蛋白质中。 萨克伊 RNA 靶标也将用于这些研究，因为它的不同之处在于 Bg1G 目标的几个方面。 我们发现在第 50 位有一个特定的突变（C 到 A 的变化） Bg1G 靶 RNA 消除了 Bg1G 识别而不改变 整体RNA结构。 我们打算寻找 Bg1G 中的突变 抑制 RNA 改变，从而希望识别接触区域。 我们还将寻找具有显性失活的 Bg1G 突变体 表型。 这些将为观察提供额外的支持 Bg1G 以二聚体形式与 RNA 结合。 最后我们将寻找突变 减轻任何显着的负面影响，目的是确定 对二聚化很重要的位点或在 二聚体。