Mechanistic Studies of the Elongation Cycle of Protein Synthesis

During translation elongation, amino acid residues are repetitively added to the growing nascent peptide chain, directed by the mRNA sequence and facilitated by two elongation factors, EF-Tu and EF-G. In this thesis, translation elongation is investigated from two new perspectives to further explore the underlying mechanisms. In the first approach, a functional EF-Tu derivative labeled with a fluorescence quencher was prepared and used for the development of two fluorescent assays to study EF-Tu:L11 and EF-Tu:tRNA FRET signals. Application of these two assays revealed the tRNA discrimination on the ribosomal A-site, the effects of error-inducing antibiotics on tRNA selection, and tRNA release from EF-Tu on the ribosome regulated by EF-Tu:tRNA affinity. In combination with other assays measuring pre-translocation complex formation, ensemble and single molecule FRET studies of the EF-Tu:L11:tRNA triangle indicated that EF-Tu:L11 separation occurs concurrently with inorganic phosphate release and tRNA accommodation, but prior to EF-Tu separation from tRNA and dissociation from the ribosome. The latter process proceeds through two possible pathways, rapid but reversible dissociation followed by a conformational change off the ribosome versus slow conformational change on the ribosome followed by rapid dissociation. Translation elongation was also studied using mRNAs specifically modified with fluorescent isomorphic nucleoside mimetics. These modified mRNAs maintain considerable functionalities in the formation of initiation, pre-translocation and post-translocation complexes, although with different efficiencies, and show fluorescence changes in different patterns upon ribosomal complex conversion. Pre-steady state measurements of these fluorescence changes demonstrate the responses of modified mRNAs to codon:anticodon interaction occurring after ternary complex initial binding to the ribosome but before tRNA accommodation into the A-site, and mRNA movements on the ribosome during translocation.