FUNCTION OF INITIATION FACTORS IN BACTERIAL TRANSLATION

细菌翻译起始因子的功能

• 批准号: 3297398
• 负责人: JOHN W HERSHEY
• 金额: $ 17.42万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3297398
• 关键词: Escherichia coli; bacterial genetics; fluorescence polarization; gene expression; genetic manipulation; genetic promoter element; genetic recombination; genetic strain; genetic translation; high performance liquid chromatography; lac operon; mutant; protein biosynthesis; protein engineering; protein structure; ribosomes; site directed mutagenesis

Protein synthesis is an integral part of gene expression, and translational control mechanisms are known to operate during the initiation phase of the process. The projects proposed here are designed to elucidate the molecular events of initiation, with special emphasis on the synthesis and mechanism of action of the three initiation factors IF1, IF2, and IF3. First, the expression of initiation factor genes in Escherichia coli will be studied by recombinant DNA techniques. The genes for IF1 (infA), IF2 (infB) and IF3 (infC) already have been cloned, mapped and sequenced. We propose to test whether or not IF1 is required for cell viability, by inactivating infA on the bacterial chromosome. Operon and protein fusion of infA and lac will be used to determine whether or not infA expression is regulated by IF1 levels (autogenous regulation) or by the stringent response. The coordinate regulation of all three initiation factor genes as a function of growth rate also will be studied in vivo will lac fusions. Overproducing strains for initiation factors will be constructed and isolation methods will be developed that employ fast protein liquid chromatography to purify the proteins. Second, we propose to study in vivo the physiological consequences of reducing the cellular levels of each of the initiation factors. In initial studies with IF2, infB was placed under control of the lac promoter/operator, and expression was regulated by the concentration of the inducer, IPTG. Strains with infA and infC under lac promoter control will be constructed and we shall study the effects of reduced factor levels on protein synthesis rates, polysome profiles, rRNA operon expression, specific proteins synthesized, and initiation pathway intermediates. Third, the mechanism of action of initiation factors will be probed by in vitro mutagenesis and by fluorescence polarization techniques. Both random and site-directed mutagenesis will be employed and the resulting mutated genes will be screened in vivo by transformation of the genes into strains carrying the chromosomal gene under lac promoter control. Growth characteristics in the absence of IPTG will depend on the mutated factor. Interesting mutant forms will be overexpressed and the mutated factor purified and studied by a variety of in vitro assays for function. Continuation of fluorescence polarization studies of factor- ribosome interactions will address quantitatively the binding of individual initiation factors to 30S and 70S initiation complexes in order to shed light on the reaction pathway of initiation. The studies should result in an increase in our knowledge of the initiation process, which is essential for understanding mechanisms of protein synthesis and translational control at the molecular level.

蛋白质合成是基因表达的组成部分， 已知平移控制机构在平移期间操作， 过程的初始阶段。 这里提出的项目是 旨在阐明启动的分子事件， 特别强调的合成和作用机制， 三个起始因子IF 1、IF 2和IF 3。 第一，表达 大肠杆菌中的起始因子基因将通过 重组DNA技术。 IF1（infA）、IF2（infB） 和IF 3（infC）已经被克隆、定位和测序。 我们建议测试IF 1是否是细胞所必需的， 通过使细菌染色体上的infA失活来提高存活力。 infA和lac的操纵子和蛋白质融合将用于 确定infA表达是否受IF 1调节 水平（自体调节）或严格的反应。 的 所有三个起始因子基因的协调调节， 生长率的功能也将在体内研究将缺乏 融合 过量产生起始因子的菌株将被 构建和隔离方法将被开发， 快速蛋白质液相色谱法纯化蛋白质。 第二、 我们建议在体内研究的生理后果 降低每种起始因子的细胞水平。 在 最初的研究与IF 2，infB被放置在控制下的紫胶 启动子/操纵子，表达受 诱导物IPTG的浓度。 具有infA和infC的菌株 在lac启动子控制下构建，我们将研究 降低因子水平对蛋白质合成速率的影响， 多核糖体谱，rRNA操纵子表达，特异蛋白 合成的和起始途径中间体。 三是 引发因子的作用机制将通过在 体外诱变和荧光偏振技术。 将采用随机和定点诱变， 将在体内筛选得到的突变基因， 将基因转化到携带染色体的菌株中 基因在lac启动子控制下。 生长特征 IPTG的缺乏将取决于突变的因子。 有趣 突变形式将被过度表达， 纯化并通过多种体外功能测定研究。 因子的荧光偏振研究的继续- 核糖体相互作用将定量地解决结合 30 S和70 S起始复合物的单个起始因子 以阐明引发的反应途径。 的 研究应该会增加我们对 启动过程，这对于理解 蛋白质合成和翻译控制的机制 分子水平。