PROBING OF INITIATION FACTOR INTERACTIONS IN TRANSLATION

University Of California Santa Cruz, Santa Cruz CA

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Abstract

Translation of mRNA into protein is thought of as occuring in three stages: initiation, elongaton, and termination. During initiation, the mRNA to be translated and the initiator tRNA are aligned on the 70S ribosome so that peptide synthesis can begin. Three protein factors (IF1, IF2, and IF3), a molecule of GTP, mRNA and fmet-tRNA are required for initiation. In this research, complexes that correspond to intermediates in the initiation pathway will be formed in vitro and probed with hydroxyl radicals generated by both free Fe(II)-EDTA and Fe(II)-EDTA tethered to unique sites on the initiation factors. The sites of protection from hydroxyl radicals and specific cleavages of the rRNA, mRNA, and tRNA will be identified by primer extension. Together, the results of these probing experiments should provide (1) a comprehensive mapping of the RNA environment of each of these factors, (2) sufficient protein-RNA interaction constraints to orient each of the factors with respect to the other components of the initiation complexes to be studied, and (3) insight into the mechanism by which the initiation factors prepare the ribosome to begin protein synthesis.