Date of Award

Summer 8-12-2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemistry

First Advisor

Barry Cooperman

Abstract

Ribosomes catalyze protein synthesis via the translation cycle, in which the translation initiation is recognized as a key step to regulate the process. The functional complexes of the bacterial ribosome undergo large conformational changes during the initiation of protein synthesis. Dramatic progress in the elucidation of ribosome structure by both X-ray crystallography and cryoelectron microscopy (cryo-EM) has provided some of the best evidence for such changes. At the same time, detailed rate studies of initiation, even though for the most part incomplete, have shown this process to be complex, multistep reactions, raising the question of the extent to which specific structural changes can be assigned to specific steps described in the proposed kinetic mechanism. By using fluorescence stopped-flow, quenched flow and FRET approaches to elucidate the kinetic mechanism of initiation, particularly the formation of a 70S initiation, we have found that following GTP hydrolysis by IF2 bound within a 70S complex, the G-domain moves toward L11-NTD, leading to increased FRET efficiency, and that Pi is released following such movement. Our results also showed that two G-proteins, IF2 and EF-Tu, can bind to the ribosome simultaneously during the transition from initiation to elongation. In vitro fluorescence assays were also developed to identify biologically active thiopeptide precursor compounds as potential new antibiotics. It is shown that some of these precursors represent novel compounds with respect to their ability to bind to ribosomes. These findings provide not only insight into the mechanism of action of thiopeptide compounds, but also demonstrate the potential of such assays for identifying novel lead compounds that might be missed using conventional inhibitory screening protocols.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Included in

Biochemistry Commons

Share

COinS