FUNCTION OF INITIATION FACTORS IN BACTERIAL TRANSLATION

细菌翻译起始因子的功能

• 批准号: 3297399
• 负责人: JOHN W HERSHEY
• 金额: $ 18.84万
• 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/3297399
• 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; recombinant DNA; 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不可或缺的一部分，而且 已知的翻译控制机制在 流程的启动阶段。这里提出的项目有 旨在阐明启动的分子事件，与 重点介绍了该化合物的合成及作用机理。 三个启动因子IF1、IF2和IF3。第一，表达 将通过以下方法研究大肠杆菌中的启动因子基因 重组DNA技术。IF1(INFA)、IF2(INFB)基因 和IF3(InfC)已被克隆、定位和测序。 我们建议测试cell是否需要IF1 生存能力，通过灭活细菌染色体上的INFA。 操纵子和蛋白融合的infa和lac将被用于 确定INFA表达是否受IF1调控 水平(自身调节)或严格的反应。这个 协调调节所有三个启动因子基因作为一个 生长速度的作用也将在体内进行研究 融合。产生启动因子的高产菌株将是 将开发构建和隔离方法，使用 快速蛋白质液相色谱分离纯化蛋白质。第二, 我们建议在体内研究阿司匹林的生理后果。 降低每种启动因子的细胞水平。在……里面 对IF2、InfB的初步研究被置于乳胶控制之下 启动子/操纵子，表达受 诱导剂IPTG的浓度。含INFA和INFC的菌株 在Lac下将构建启动子控制，我们将研究 降低因子水平对蛋白质合成速率的影响， 多聚体图谱、rRNA操纵子表达、特定蛋白 合成和启动途径中间体。第三， 引发因子的作用机制将通过INS来探讨 体外诱变和荧光偏振技术。 随机和定点诱变都将被使用，并 由此产生的突变基因将通过以下方式在体内进行筛选 将基因转化为携带染色体的菌株 在lac启动子控制下的基因。林木的生长发育特征 IPTG的缺失将取决于突变因素。有意思的 突变形式会过度表达，突变因子 通过各种体外试验对其进行纯化和研究。 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 对30S和70S起爆复合体的个别起爆因素 以阐明引发反应的途径。这个 研究应该会增加我们对生物多样性的认识 启蒙过程，这是理解 蛋白质合成和翻译调控的机制 分子水平。