Transfer rna dynamics and transfer-messenger rna accommodation in bacterial ribosomes at the single-molecule level
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چکیده
TRANSFER RNA DYNAMICS AND TRANSFER-MESSENGER RNAACCOMMODATION IN BACTERIAL RIBOSOMES AT THE SINGLE-MOLECULELEVEL byMAY DAHERDecember 2012Advisor: Dr. David RuedaMajor: Chemistry (Biochemistry)Degree: Doctor of PhilosophySingle-molecule spectroscopy, protein-induced fluorescence enhancement(PIFE), fluorescence resonance energy transfer (FRET), and several biochemical toolswere applied to study transfer RNA (tRNA) dynamics and transfer-messenger RNA(tmRNA) accommodation inside ribosomes. In the first project of this thesis work,structural characterization of the tRNA-like domain of tmRNA (TLD) in complex withSmpB protein was carried out, and the results reveal no change in the globalconformation or the flexibility of the TLD upon SmpB binding. In contrast, magnesiumions induce a compaction of the TLD structure, suggesting that flexibility in the H2astem of TLD may allow different conformations of tmRNA, as the TLD and mRNA-likedomain (MLD) need to be positioned differently while moving through the ribosome. Inthe second project, an assay was developed to study the dynamics of tRNA and tmRNAaccommodation inside ribosomes. The results from this project reveal fluorescenceintensity changes of a dye-labeled tRNA (Cy3-tRNA) because of changes in the
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