Molecular Basis of Ribosomal Frameshifting

核糖体是一种复杂的细胞机制，负责促进mRNA指导的翻译 在每个活着的有机体中产生所有蛋白质的遗传密码。核糖体必须选择正确的tRNA来 对mrna进行解码，促进多肽键的形成，然后将tRNAs通过其三种功能移动。 以动态和精心安排的方式区分不同的tRNA结合位点。与此相关的错误 蛋白质合成的过程对基因表达和细胞功能都是有害的。因此， 核糖体对通用的mRNA三核苷酸密码(或“阅读框架”)的准确维持是 关键，但关于这是如何被控制的，或者如何以编程的方式偏离的分子细节，并不是很好 明白了。信使核糖核酸框架维持的机制及其与转录因子和信使核糖核酸的相互作用 合作防止信使核糖核酸的转移仍是未知的。我们的初步数据显示，移码 抑制子tRNA导致+1帧将它们的偏向位置移向E位，解码后拉出 30S头部结构域与延伸因子结合不相容。在目标1中，我们将确定内生性 位于第37位的tRNA修饰调节着mRNA的框架。接下来，我们将确定延伸率如何 识别正在进行移码的核糖体复合体的因子以及这些因子是否起到调节作用 在mRNA框架维护中的作用。在目标3中，我们将确定启动子tRNAfMet和启动因子是如何 协作维护信使核糖核酸AUG起始框架。在目标4中，我们将研究两个不同的mRNA控制 基因表达。这些目标将通过结合以下结构生物学来实现 大的功能性核糖体复合体和生化方法，如smFRET和RNA形状探测。

Ribosomes are the complex, cellular machinery responsible for promoting mRNA-directed translation of the genetic code to produce all proteins in every living organism. The ribosome must select correct tRNAs to decode the mRNA, facilitate peptide bond formation, and then move the tRNAs through its three functionally distinct tRNA binding sites in a dynamic and exquisitely orchestrated manner. Errors associated with this process of protein synthesis are detrimental to gene expression and hence cellular function. Therefore, accurate maintenance of the universal mRNA three-nucleotide code (or “reading frame”) by the ribosome is critical but the molecular details of how this is controlled, or deviated from in a programmed manner, is not well understood. The mechanism of mRNA frame maintenance and how RNAs including tRNAs and mRNA collaborate to prevent mRNA shifting remains unknown. Our preliminary data reveals that frameshift suppressor tRNAs cause +1 frameshifting their biased position towards the E site post decoding that pulls the 30S head domain incompatible with elongation factor binding. In Aim 1, we will determine how endogenous tRNA modifications located at position 37 regulate the mRNA frame. Next, we will determine how elongation factors recognize a ribosome complex undergoing frameshifting and whether these factors play regulatory roles in mRNA frame maintenance. In Aim 3 we will identify how initiator tRNAfMet and initiation factors collaborate to maintain the mRNA AUG start frame. In Aim 4, we will study two different mRNAs that control gene expression. Together these aims will be accomplished through a combination of structural biology of large, functional ribosomal complexes and biochemical methods such as smFRET and RNA SHAPE probing.