Regulation of Ribosome Structural Dynamics by Antibiotics, Translation Factors and mRNA Secondary Structure

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2017.The structural dynamics of the ribosome underlie the mechanism by which information encoded in messenger RNAs is translated into a polypeptide chain. During protein synthesis, the small and large ribosome subunits rotate relative to each other while the L1 stalk, a mobile domain of large subunit, moves inward/outward relative to the core of the large subunit. Translation factors, antibiotics and structured RNAs are able to regulate or perturb translation by modulating ribosome structural dynamics. Furthermore, translation factors also undergo large structural rearrangements while interacting with the ribosome. Using single-molecule Förster resonance energy transfer (smFRET) to follow the structural rearrangements of the ribosome and translation factors, I have found (a) that the antibiotic, blastidicin S (BlaS), slows down intersubunit rotation, (b) the HIV and dnaX frameshift stimulating stemloops inhibit ribosome translocation by blocking tRNA binding to the A site of the ribosome, and (c) bacterial initiation factor 2 (IF2) positions ribosome subunits in a semi-rotated orientation during the subunit-joining step of translation initiation. Many small molecule antibiotics that target translation perturb ribosome structural dynamics. BlaS is a potent translation inhibitor in both eukaryotes and bacteria; however, the mechanism of BlaS action was not clear. I found that BlaS interacts with the P-site tRNA and inhibits spontaneous intersubunit rotation in bacterial ribosomes. Further studies by our collaborators from UMASS School of Medicine revealed that BlaS targets proteins synthesis via a unique mechanism hampering translation termination. During translation elongation, the ribosome moves along mRNA in a codon-by- codon manner and unwinds mRNA secondary structure. However, specific mRNA stemloops, such as the frameshift stimulating signal (FSS) from the E. coli dnaX gene, induce ribosome stalling and frameshifting. We sought to determine how FSS RNA stemloops stall ribosome translocation despite efficient helicase activity of the ribosome. Surprisingly, we found that the FSSs from HIV and dnaX mRNAs block tRNA binding to the A site of the ribosome, thereby inhibiting translocation mediated by the bacterial ribosome translocase, elongation factor-G. Our future studies will identify properties of mRNA secondary structure that inhibit A-site tRNA binding. Thus, our studies will provide fundamental insights into the regulation of translation elongation by mRNA structure and the mechanism of programmed ribosome frameshifting. We also extended our studies beyond translation elongation to elucidate the molecular mechanism of the subunit-joining step of translation initiation in bacteria. Initiation of protein synthesis is the key regulatory step of translation. In bacteria, translation initiation is controlled by initiation factors (IFs) 1, 2 and 3, which ensure selection of the initiator tRNA and promote joining of the large and small ribosomal subunits. Using smFRET, we find that IF2, a translational GTPase, transiently stabilizes the ribosome in a distinct conformation where the ribosomal subunits are in a semi- rotated orientation and the L1 stalk is in a half-closed position (3). Our results suggest that positioning subunits in a semi-rotated orientation facilitates subunit association and support a model in which L1 stalk movement is coupled to intersubunit rotation and/or IF2 binding