Ribosomal translocation in real time : Method development to Applications

Translational elongation is the process in which the ribosome adds one amino acid at a time to the nascent peptide chain. As the ribosome elongates the peptide chain by 14 - 20 amino acids per second and performs hundreds of such cycles per protein, ‘elongation’ is one of the most crucial steps in translation. During elongation, the ribosome must move precisely by one codon along the mRNA after peptidyl transfer. This step is known as ‘ribosomal translocation’, which is catalyzed by the elongation factor G (EF-G). Despite extensive research, the exact sequence of events in translocation is still unclear. Thus, development of an in vitro assay, which would allow precise kinetic measurement of the steps involved in ribosomal translocation, is highly important. In 2003, a research group led by Simpson Joseph designed a fluorescence-based assay to monitor translocation in stopped-flow using a short mRNA labeled with fluorescent-dye pyrene at the 3’ end. Although optimized for the highest fluorescence change, this assay showed significantly slower translocation than what has been measured by conventional translocation assays. Moreover, when performed with a popular peptidyl tRNA analog, it produced complex multiphasic kinetics. Therefore, this assay was not only limited for deriving physiologically relevant rates, but also the analysis of the rates from the kinetic data was challenging. In this thesis, we have optimized the fluorescent-mRNA based stopped-flow translocation assay, by carefully calibrating it with the functional tripeptide formation assay performed in quench-flow. First, we identified the most suitable mRNA length that is optimal for both the fluorescence signal and the rate of translocation. Further, by systematically varying temperature, Mg2+, EF-G and tRNA analog concentration, we have derived the ideal condition for obtaining near monophasic kinetics in stopped-flow, which allows determination of the translocation rate in an unambiguous manner. This optimized assay has further been tested in different contexts involving translocation, which include (i) characterization of the GTPase deficient EF-G mutants, (ii) studying the effect of non-hydrolyzable GTP analogs, (iii) evaluating the effect of extension of the C-terminal tail of the ribosomal protein S13 in the decoding center, and (iv) understanding the mechanism of action of a novel aminoglycoside antibiotic ‘arbekacin’ in translation. These studies altogether provide deep understanding for how different factors can modulate ribosomal translocation