Farrell, Ian S

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  • Publication
    Investigating tRNA Release from the Bacterial Ribosome
    (2011-08-12) Farrell, Ian S
    Translation of mRNA into proteins is integral in all living organisms, and takes place on the ribosome. In recent years, the X-ray crystal structures of biologically relevant ribosome complexes came into light, and the advance of kinetic studies was soon to follow, leading to a better understanding of the general ribosomal mechanism. However, there still remains some ambiguity in certain ribosome functions. Ribosomal protein L1 initially became relevant in the early 1980s when it was determined that ribosomes lacking L1 showed a decreased capacity for in vitro protein synthesis. Later, it was shown that the L1-stalk is a highly mobile region of the ribosome, and that it may be involved in the release of deacylated-tRNA from the exit-site, after translocation. By using a fluorescently labeled L1 reconstituted ribosome as an E-site probe we were able to study the release of deacylated-tRNA from translocating ribosomes in a time-resolved manner. The movement of the L1-stalk with relation to the deacylated-tRNA was measured using fluorescence resonance energy transfer (FRET) measurements between labeled L1 and labeled tRNAs (L/t FRET). Further, the movement of deacylated-tRNA with relation to the P-site tRNA was measured using fluorescently labeled tRNAs (t/t FRET), and the release of deacylated-tRNA was measured using changes in anisotropy. We demonstrate that the deacylated-tRNA can be released from the ribosome via three possible different pathways, depending on the conditions. Further, in Chapter V, we begin to demystify the interaction that the E-site region of the ribosome has with deacylated-tRNAs in solution, and demonstrate the changes on deacylated-tRNA release when excess tRNAs are present. The optimization of the creation of a viable E-site probe will prove to be important for future studies in both kinetic work and single molecule work when focusing on tRNA interaction with the E-site of the ribosome.