TRANSLATIONAL ATTENUATION AS A GENE REGULATOR

翻译衰减作为基因调节因子

• 批准号: 3301867
• 负责人: PAUL S LOVETT
• 金额: $ 22.43万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE COUNTY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3301867
• 关键词: Bacillus subtilis; RNA; acetyl coA acetyltransferase; chemical cleavage; chloramphenicol; endonuclease; enzyme induction /repression; gene expression; genetic manipulation; genetic regulation; genetic transcription; genetic translation; gram positive bacteria; nucleic acid structure

Chloramphenicol (Cm) inhibits bacterial growth by blocking peptide elongation during translation on 70S ribosomes. Cm also acts as an inducer of the expression of cat genes (specify chloramphenicol acetyltransferase) in Gram-positive bacteria. Induction of one gene, cat-86, has been shown to e due to activation of mRNA translation. This results from the ability of Cm to stall ribosomes translating a cat-86 RNA stem-loop that sequesters the cat-86 ribosome binding site. The long term goal of this proposal is to explain the mechanism which allows Cm to activate cat-86, and other inducible cat genes. It is also expected that these studies will show the breadth of the principles of the attenuation regulatory model in controlling gene expression. By use of site directed mutagenesis and recombinant DNA technology it is now possible to determine whether the proposed Cm stall site is an mRNA or protein sequence, and the sequence of the site. The special relationship between the stall site and the regulated RNA secondary structure will be identified. Uncommon examples of apparently Cm- insensitive translation exist in bacteria. Do these result from the fortuitous absence of a Cm stall site? Erythromycin probably stalls ribosomes at a site different from Cm. It has been possible to convert cat-86 to an erythromycin inducible gene, and this should provide a novel approach to identify the presumptive erythromycin stall site. cat-86 mRNA is cleaved at two locations by an apparent endonuclease in B. subtilis. We believe we have identified one of the sites susceptible to cleavage. The sequence this site is similar to exon-intron junctions in eukaryotic mRNA. Experiments to characterize this endonuclease activity are proposed. An inducible cat gene from a gram-negative and an inducible streptomycin- resistance gene from a gram-positive will be characterized with respect to the induction mechanisms.

氯霉素 (Cm) 通过阻断肽来抑制细菌生长 70S 核糖体翻译过程中的延伸。 Cm 还充当 猫基因表达诱导剂（指定氯霉素 乙酰转移酶）存在于革兰氏阳性菌中。 一个基因的诱导， cat-86，由于 mRNA 翻译的激活而被证明是 e。 这 来自 Cm 阻止核糖体翻译 cat-86 的能力的结果 隔离 cat-86 核糖体结合位点的 RNA 茎环。 长的 该提案的长期目标是解释允许 Cm 的机制 激活 cat-86 和其他诱导型猫基因。 还预计 这些研究将展示衰减原理的广度 控制基因表达的调控模型。 通过使用定点诱变和重组 DNA 技术， 现在可以确定提议的 Cm 失速位点是否是 mRNA 或蛋白质序列，以及位点的序列。 特别的 失速位点与受调控RNA二级之间的关系 结构将被识别。 明显 Cm- 的不常见例子 细菌中存在不敏感翻译。 这些结果是否源于 偶然没有 Cm 摊位站点？ 红霉素可能会停滞 核糖体位于与 Cm 不同的位点。 已经可以转换了 cat-86 到红霉素诱导基因，这应该提供一种新的 确定假定的红霉素失速位点的方法。 cat-86 mRNA 在 B. 枯草芽孢杆菌。 我们相信我们已经确定了易受影响的站点之一 分裂。 该位点的序列类似于外显子-内含子连接 真核mRNA。 表征该核酸内切酶活性的实验 被提议。 来自革兰氏阴性菌的诱导型猫基因和诱导型链霉素- 革兰氏阳性菌的抗性基因将受到尊重 到诱导机制。