| Translation of genetic information into functional protein involves a mutli-step process carried out by the large ribonucleoprotein complex known as the ribosome. Following peptide bond formation the interaction of elongation factor G (EF-G) and the energy from GTP hydrolysis are normally required to promote accurate translocation of the tRNA:mRNA complex through the cavity of the ribosome, allowing engagement of the next downstream codon. While there has been considerable focus on EF-G function, the energy barriers within the ribosome that maintain a stable pre-translocation state are not well understood. Experiments in the 1970s demonstrated that, under certain conditions, synthesis of poly-phenylalanine from a poly-uridine template can occur in the absence of EF-G and other enzymatic factors, suggesting that the mechanism of translocation is inherent to the structures of the ribosome and bound tRNA:mRNA complex. The following dissertation focuses on EF-G independent translocation and identifies novel ribosomal components critical in movement of the tRNA:mRNA complex. Utilizing previously discovered techniques of thiol modification of the ribosome, we have established that EF-G independent translocation is the result of authentic three nucleotide movement down the mRNA. Mutagenesis and protein deficiency studies using in vitro reconstituted 30S particles reveal that the small subunit proteins, S12 and S13, are critical in stabilizing the pre-translocation state and lead to a model in which the interface proteins act to regulate the underlying RNA-based translocation machinery. Additionally, a novel in vitro selection method involving a photo-activatable crosslink in tRNAVal was developed for the identification of bases in 16S rRNA that are involved in the movements of translocation. Finally, other work focuses on the reactivity of the peptidyl transferase center of the ribosome in the pre- and post-translocation states and further establishes that following peptide bond formation the ribosome must sample or reside in an intermediate state of tRNA binding independent of the action of EF-G. |
