Open Access Dissertation
Date of Award
Single-molecule spectroscopy, protein-induced fluorescence enhancement (PIFE), fluorescence resonance energy transfer (FRET), and several biochemical tools were applied to study transfer RNA (tRNA) dynamics and transfer-messenger RNA (tmRNA) accommodation inside ribosomes. In the first project of this thesis work, structural characterization of the tRNA-like domain of tmRNA (TLD) in complex with SmpB protein was carried out, and the results reveal no change in the global conformation or the flexibility of the TLD upon SmpB binding. In contrast, magnesium ions induce a compaction of the TLD structure, suggesting that flexibility in the H2a stem of TLD may allow different conformations of tmRNA, as the TLD and mRNA-like domain (MLD) need to be positioned differently while moving through the ribosome. In the second project, an assay was developed to study the dynamics of tRNA and tmRNA accommodation inside ribosomes. The results from this project reveal fluorescence intensity changes of a dye-labeled tRNA (Cy3-tRNA) because of changes in the fluorophore environment. These changes are sensitive to both magnesium concentration and the presence of antibiotics. Interestingly, these changes can be correlated to spontaneous ribosomal ratcheting. Monitoring tmRNA entrance and accommodation was also studied at the single-molecule level. The data reveal that the accommodation of tmRNA occurs in a multistep process, in which three major FRET states are present. The low FRET state might represent the initial binding of tmRNA. The mid and high FRET states are believed to represent the pre-accommodation and full accommodation, respectively, of tmRNA inside the ribosome. These states were successfully assigned by using specific antibiotics that allow stalling at each FRET state. Overall, our data suggest that the accommodation of tmRNA occurs in the same manner as a canonical tRNA. These results have allowed the successful dissection of the first step of the tmRNA task inside ribosomes, which is important for understanding further steps in the pathway and unraveling the mystery of this RNA function. Such results could be beneficial toward developing antibiotics that are urgently needed for targeting pathogenic bacteria.
Farhat, May Daher, "Transfer rna dynamics and transfer-messenger rna accommodation in bacterial ribosomes at the single-molecule level" (2012). Wayne State University Dissertations. 565.