Molecular Basis of Ribosomal Frameshifting

核糖体是复杂的细胞机器，负责促进mRNA指导的翻译， 基因编码来生产所有生物体内的蛋白质。核糖体必须选择正确的tRNA， 解码mRNA，促进肽键的形成，然后通过其三个功能区移动tRNA， 不同的tRNA结合位点，以动态和精致的协调方式。与此相关的错误 蛋白质合成过程对基因表达和细胞功能有害。因此，我们建议， 核糖体对通用mRNA三核苷酸密码（或“阅读框架”）的精确维护， 关键的，但这是如何控制的分子细节，或偏离程序的方式，是不是很好， 明白mRNA框架维持的机制以及RNA（包括tRNA和mRNA）如何维持框架的机制 合作防止mRNA转移仍然是未知的。我们的初步数据显示移码 抑制性tRNA在解码后使其偏向位置向E位点+1移码， 30S头部结构域与延伸因子结合不相容。在目标1中，我们将确定内源性 位于位置37的tRNA修饰调节mRNA框架。接下来，我们将确定伸长率 因子识别正在进行移码的核糖体复合物，以及这些因子是否起调节作用。 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 协作以维持mRNA AUG起始框架。在目标4中，我们将研究两种不同的mRNA， 基因表达。总之，这些目标将通过结构生物学的结合来实现， 大的功能性核糖体复合物和生物化学方法，如smFRET和RNA SHAPE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.