Structure-function relationship between multiple translation initiation factor 3

描述（由申请人提供）：最近在蓝藻Fremyella diplosiphon中发现了两种翻译起始因子IF 3，IF 3a和IF 3b，这为更好地理解这些蛋白质如何在转录衰减和翻译水平上调节基因表达提供了令人兴奋的长期机会。IF 3是翻译起始过程的重要组成部分，因此在许多细菌如大肠杆菌中细胞存活。然而，遗传筛选显示F. diplosiphon对于生存力不是必需的，并且对其测序的基因组的分析揭示了编码IF 3（IF 3b）的第二个基因。此外，IF 3a和IF 3b都是E.大肠杆菌菌株缺乏IF 3，表明两者都是功能性IF 3蛋白。此外，IF 3a在转录后水平调节cpeC操纵子的转录物的丰度，以响应环境光颜色的变化。 为了了解这两个IF 3s在翻译调控中的作用，我建议首先使用野生型菌株，缺乏IF 3a的菌株和缺乏IF 3b的菌株的核糖体分析，以确定在每个菌株中被翻译的mRNA。此外，我建议使用深度测序的总mRNA在每个菌株中研究IF 3a和IF 3b在全基因组调节转录丰度在这种生物体中的可能作用。综合分析这些菌株的整体翻译活性和转录水平，将提供一个全面的看法，IF 3a和IF 3b如何影响中央细胞过程中的F。双虹吸管这些功能性研究将得到结构性研究的补充。IF 3a和IF 3b在这种生物体中的存在提供了一个独特的机会，使用X射线晶体学和/或NMR对其结构进行比较分析，以将任何结构差异与功能分析中发现的差异联系起来。总的来说，这项研究将为这类重要蛋白质的结构和功能之间的关系提供重要的见解，这些结果不可能从任何其他含有单一必需IF 3的真细菌中获得。 翻译是一个重要的细胞过程，我的研究结果将扩大我们对IF 3蛋白在细菌翻译起始中的作用的理解。由于IF 3已知是细菌中的一种必需蛋白质，并且其结构与其等效的真核蛋白质eIF 1不同，因此研究IF 3功能与其结构的关系有可能导致新型抗生素的开发，随着越来越多的细菌对已知抗生素产生耐药性，这一点非常重要。

DESCRIPTION (provided by applicant): The recent discovery of two translation initiation factor IF3s, IF3a and IF3b, in the cyanobacterium Fremyella diplosiphon provides an exciting long-term opportunity to better understand how these proteins regulate gene expression at the level of transcriptional attenuation and translation. IF3s are an essential component of the translation initiation process, and therefore cell survival, in many bacteria such as Escherichia coli. However, a genetic screen has revealed that IF3a in F. diplosiphon is not essential for viability, and analysis of its sequenced genome uncovered a second gene encoding an IF3 (IF3b). In addition, both IF3a and IF3b complement an E. coli strain lacking IF3, indicating that both are functional IF3 proteins. Furthermore, IF3a regulates the abundance of transcripts from the cpeC operon at the post-transcriptional level in response to changes in ambient light color. To understand the role of these two IF3s in translational regulation, I propose to initially use ribosome profiling of wild-type strains, strains lacking IF3a, and strains lacking IF3b to identify the mRNAs that are being translated in each strain. In addition, I propose to use deep sequencing of the total mRNA in each strain to study the possible roles of IF3a and IF3b in the genome-wide regulation of transcript abundance in this organism. The combined analyses of global translational activity and transcript levels in these strains will provide a comprehensive view of how IF3a and IF3b affect central cellular processes within F. diplosiphon. These functional studies will be complemented by structural studies. The existence of IF3a and IF3b in this organism provides a unique opportunity to conduct a comparative analysis of their structures using X-ray crystallography and/or NMR to relate any structural differences to differences uncovered in the functional analysis. Overall, this research will provide important insights into the relationship between structure and function for this important class of proteins, results that have not been possible to obtain from any other group of eubacteria, which contain a single, essential IF3. Translation is an important cellular process, and the results of my research will expand our understanding the role of the protein IF3 in bacterial translation initiation. Because IF3 is known to be an essential protein in bacteria and has a structure that is distinct from its equivalent eukaryotic protein, called eIF1, the study of how IF3 function relates to its structure has the potential to lead to the development of novel antibiotics, which is important as more bacteria become resistant to known antibiotics.