Project Summary/Abstract 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.