Title: The Role of Ribosome and tRNA Dynamics in the Regulation of Translation Elongation
Description: Protein synthesis, one of nature's most fundamental processes within all living cells, is catalyzed by the ribosome, a highly conserved, massive, two-subunit ribonucleoprotein complex. Ribosomes synthesize proteins based on the sequence of triplet-nucleotide codons presented by the messenger RNA (mRNA) template, using aminoacyl-transfer RNAs (aa-tRNAs) substrates, which deliver individual amino acids to the ribosome. Recent biochemical, structural, dynamic and computational studies have uncovered large-scale conformational changes of the ribosome, its tRNA substrates, and translation factors that play important roles in regulating protein synthesis, especially during the elongation phase of translation. For example, translocation of the ribosome along its mRNA template involves several conformational rearrangements of the ribosomal pre-translocation (PRE) complex, including the rotation of two ribosomal subunits, closure of the L1 stalk element, and reconfigurations of the ribosome-bound tRNAs.

Importantly, modulation of these conformational changes of PRE complexes is used as a strategy by the cell and ribosome-targeting antibiotics to regulate translation elongation. Therefore, a complete understanding of the conformational dynamics of ribosomal complexes will not only improve our knowledge on how translation is regulated, but also provide crucial information for designing next-generation antibiotics. This thesis presents efforts demonstrating several strategies the cell develops in order to regulate translation by modulating the conformational dynamics of ribosomal complexes. In Chapter 2, I investigate if and how the individual dynamics of intersubunit motion, tRNA and L1 stalk are coordinated within PRE complexes, so that the translocation reaction is facilitated. To address this question, the dynamics of ribosomal intersubunit rotation were predictably perturbed using either structurally guided ribosome mutagenesis as well as an ribosome-targeting antibiotic translation inhibitor.

Correspondingly, I used two single-molecule fluorescence resonance energy transfer (smFRET) signals to directly monitor how perturbation of the dynamics of intersubunit rotation alter the dynamics of P-site tRNA and the L1 stalk in PRE complexes. Taken together with the results of my complementary in vitro biochemical assays, my smFRET work clearly demonstrates that the ribosome coordinates individual conformational changes to maximize and regulate the efficiency of the translocation reaction. It is very likely that this strategy is used by the ribosome in other steps during translation for efficient chemical or mechanical reactions, and is taken advantage of by translation factors and antibiotics as part of the mechanisms through which they regulate and inhibit translation, respectively. Energy-dependent translational throttle A (EttA) is one regulatory translation factor that has been recently discovered and characterized through a collaboration between the Hunt, Gonzalez, and Frank laboratories (Chapter 3).

Biochemical experiments have shown that in the presence of a high ADP/ATP ratio, EttA inhibits formation of the first peptide bond, and such inhibition is relieved upon addition of ATP, indicating that EttA may regulate the synthesis of proteins in response to the energetic status of the cell, as reflected by the cellular ADP/ATP ratio. Complementary cryo-EM studies have shown that the ATP-bound form of EttA binds to the ribosome at the E-site from where it directly contacts and forms bridging interaction between the L1 stalk and P-site tRNA. The results of my smFRET experiments demonstrate that EttA differentially modulates the conformation and/or dynamics the L1 stalk, depending on whether EttA is bound to ADP or ATP, thereby providing a possible rationale for the distinct effects of EttA on dipeptide synthesis in the presence of ADP vs. ATP. My smFRET data, together with the biochemical and structural efforts, demonstrate that EttA functions, at least in part, by restricting ribosome and tRNA dynamics that are crucial for translation.

More importantly, our data support a model for the interaction of EttA with the ribosomal complex and its regulation of translation at the start of the elongation cycle, the molecular mechanism of which EttA uses has never been found among all other known translational regulatory factors. +1 non-programmed ribosomal frameshifting (+1 FS), in which the elongating ribosome slips by one nucleotide towards the 3' end of the mRNA during translation, occurs at a low frequency as a translational error. Proline-tRNA with an anticodon GGG (tRNAProGGG) is prone to induce +1 FS, and the evolution of post-transcriptional modifications of tRNAProGGG is used as a strategy by nature to suppress this error. However, the mechanism underlying this suppression has not been previously characterized. tRNAProGGG modifications have been shown to play important roles in regulating the conformational stability and flexibility of the secondary and tertiary structures of tRNAs and therefore have the potential to regulate the conformational dynamics of ribosomal complexes during translation elongation.

However, the effect of these modifications on elongating ribosomal complex dynamics is completely unknown, thus greatly impeding our understanding of the role that they play in maintaining the translational reading frame. Chapter 4 presents the efforts to elucidate the mechanism by which tRNA modifications suppress +1 FS by investigating the effects that modifications of tRNAProGGG have on regulating the conformational dynamics of ribosomal complexes in a collaboration with the Hou group at Thomas Jefferson University. The preliminary results from my smFRET experiments suggest that tRNAProGGG modifications do indeed play a role in modulating the dynamics of ribosomal complexes. More importantly, the combination of tRNA without modifications and mRNA carrying a sequence that is prone to induce +1 FS dramatically alters ribosome dynamics, probably by affecting tRNA flexibility, tRNA-ribosome, tRNA-mRNA, and mRNA-ribosome interactions, all of which could have important implications for how +1 FS occurs.