Structure-function relationship between multiple translation initiation factor 3

描述（由申请人提供）：最近在蓝藻白虹菌中发现了两个翻译起始因子IF3s， IF3a和IF3b，为更好地了解这些蛋白如何在转录衰减和翻译水平上调节基因表达提供了一个令人兴奋的长期机会。在大肠杆菌等许多细菌中，IF3s是翻译起始过程的重要组成部分，因此是细胞存活的重要组成部分。然而，基因筛选显示，F. diplosiphon中的IF3a对生存能力并不是必需的，并且对其测序基因组的分析发现了第二个编码IF3的基因（IF3b）。此外，IF3a和IF3b都补充了缺乏IF3的大肠杆菌菌株，表明它们都是功能性的IF3蛋白。此外，IF3a在转录后水平上调节cpeC操纵子转录本的丰度，以响应环境光色的变化。为了了解这两种IF3s在翻译调控中的作用，我建议首先使用野生型菌株、缺乏IF3a的菌株和缺乏IF3b的菌株的核糖体分析来确定每种菌株中正在翻译的mrna。此外，我建议对每个菌株的总mRNA进行深度测序，研究IF3a和IF3b在该生物体全基因组转录物丰度调控中的可能作用。这些菌株的全球翻译活性和转录水平的综合分析将提供IF3a和IF3b如何影响F. diplosiphon中心细胞过程的全面观点。这些功能研究将由结构研究补充。IF3a和IF3b在这种生物体中的存在提供了一个独特的机会，可以使用x射线晶体学和/或核磁共振对它们的结构进行比较分析，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，这些结果是不可能从其他任何一类真细菌中获得的，这些真细菌含有单一的，必需的IF3。翻译是一个重要的细胞过程，我的研究结果将扩大我们对蛋白质IF3在细菌翻译起始中的作用的理解。由于已知IF3是细菌中的一种必需蛋白质，其结构不同于其等效的真核蛋白eIF1，因此对IF3功能与其结构的关系的研究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，这一点很重要。

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. PUBLIC HEALTH RELEVANCE: Translation is an essential 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 many 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.