The ribosomal scanning model postulates a three-step mechanism for initiation codon selection. 43S preinitiation complexes comprising 40S subunits, initiator tRNA and initiation factors (elF) elFs 2, 3, 1 and 1A first bind to the 5'-end of mRNA and then scan downstream along the 5'-untranslated region (5'-UTR) until they encounter the first AUG triplet in a favorable context. The scanning mechanism requires continuous protection against partial basepairing between triplets in the 5'-UTR and the anticodon of initiator tRNA. A key role in maintaining the fidelity of initiation codon selection belongs to elF1, which enables 43S complexes to reject codon-anticodon mismatches, to recognize the context of the initiation codon and to discriminate against AUG triplets that are <8 nt. from the 5'-end of mRNA. The position of elF1 on the 40S subunit suggests that elF1 performs its function indirectly by inducing conformational changes in the initiation complex. To investigate the mechanism of eIFl's action we shall (i) study conformational changes in initiation complexes by determining the influence of elF1 on the position of mRNA on the 40S subunit, (ii) analyze the mechanism of dissociation of aberrant ribosomal complexes by elF1, and (iii) investigate elFl's involvement in selection of initiator tRNA. To get insights into evolutionary origin of elF1 we shall determine the activities of its prokaryotic structural and functional homologues, YciH and initiation factor IF3, respectively, in both prokaryotic and eukaryotic translation initiation. To provide the basis for understanding the molecular mechanism of ribosomal scanning and initiation codon recognition we shall apply a powerful combination of biophysical (cryo-electron microscopy, hot tritium bombardment) and biochemical (UV cross-linking, foot-printing and hydroxyl radical cleavage) techniques to obtain a well-defined architectural model of 43/48S ribosomal complexes. The resulting model will establish the relative positions of initiation factors, initiator tRNA and mRNA on the 40S subunit, and will reveal the structure of mRNA threading channel.
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