Title: Regulation of Release Factor 2 in Non-canonical Translation Pathways
Description: Protein synthesis, or translation, is a complex, multi-step process that requires regulatory and quality control mechanisms to ensure the accurate production of proteins. Two major challenges during bacterial protein synthesis are maintaining the accuracy of translation during the elongation stage and resolving stalled ribosomal complexes. Interestingly, bacteria have evolved two mechanisms, a post-peptidyl transfer quality control (post PT QC) and a ribosome rescue mechanism, to counter these challenges. Both of these mechanisms make use of a protein factor that normally functions during translation termination, Release Factor 2 (RF2), along with an additional protein factor, Release Factor 3 (RF3) for post PT QC and Alternative ribosome-rescue factor A (ArfA) for ribosome rescue, to achieve these non-canonical functions. The mechanistic role of RF3 and ArfA in these two pathways remains unclear; however, they may play a role in regulating RF2 in context of these non-canonical pathways.

As a step toward understanding the role of RF3 and ArfA in post PT QC and ribosome rescue and, in particular, their role in the regulation of RF2, I sought to determine the effect of RF3 and ArfA on the binding kinetics of RF2 in post PT QC and ribosome rescue pathways. Using a single-molecule fluorescence resonance energy transfer (smFRET) signal between the P-site peptidyl-tRNA and RF2, the binding and dissociation of RF2 can be directly monitored in the absence or in the presence of RF3 or ArfA. In Chapter 2, I describe the development of smFRET signals using different chromophores, cyanine 3 to cyanine 5 (Cy3-Cy5) or to a fluorescence quencher (Cy3-QSY9). The Cy3-Cy5 and Cy3-QSY9 smFRET signals complement each other for monitoring RF2 binding; whereas Cy3-Cy5 is suitable for observing stable binding using low substrate concentration, Cy3-QSY9 is suitable for observing transient binding using high substrate concentration. The RF2 binding and dissociation to ribosomal complexes was first examined in the absence of other factors thus providing the foundation for studying the regulation of RF2 binding by RF3 or ArfA.

In the bacterial post PT QC mechanism, RF3 enhances the rate of RF2-mediated peptide release to catalyze premature termination of miscoded protein, thus ultimately increasing the fidelity of protein synthesis1. Without addition of RF3, the rate of RF2-mediated peptide release is too slow to compete with the rate of protein synthesis. In Chapter 3, the role of RF3 on RF2 binding kinetics in post PT QC was investigated using both an fMet-Lys-tRNALys(Cy3) to RF2(Cy5) smFRET signal and an fMet-Lys-tRNALys(Cy3) to RF2(QSY9) smFRET signal. The ArfA-RF2 ribosome rescue pathway is a backup mechanism for trans-translation, which relieves stalled ribosomal complexes by providing an open reading frame coding for both a degradation tag and a stop codon2. Because the expression of ArfA is under strict control by trans-translation, the ArfA-RF2 pathway only functions in the absence of active trans-translation. More importantly, deletion of both the trans-translation and ArfA-RF2 pathways leads to synthetic lethality in E. coli, highlighting the critical role of ribosome rescue in vivo3.

In Chapter 4, I used an fMet-Phe-tRNAPhe(Cy3) to RF2(Cy5) smFRET signal to evaluate the role of ArfA on RF2 binding and dissociation in the ribosome rescue pathway. Collectively, these studies survey the regulation of RF2 binding kinetics by RF3 or ArfA in performing non-canonical functions such as post PT QC and ribosome rescue in bacteria.