Translation initiation requires eukaryotic initiation factors (eIFs) 2 and 3 to bind initiator tRNA to a 40S ribosomal subunit and eIFs 4A, 4B and 4F to attach the resulting 43S complex to the 5' end of an mRNA. In a reaction reconstituted in vitro from purified translation components, this complex did not reach the beta-globin initiation codon without eIFs 1 and 1A.
The aim of this proposal is to determine how the 43S complex scans from the 5' end to the initiation codon and how it is then joined to a 60S subunit to form an active 80S ribosome. We shall use mutant mRNAs to verify that initiation in these reactions meets experimental criteria for scanning and initiation codon recognition. If additional activities are required, necessary factors will be purified from permissive cell extracts in conjunction with appropriate assays of function. The same strategy will be used to purify and identify factors required for subunit joining. Ribosomal scanning will then be assayed directly using fluorescence resonance energy transfer to determine the proximity of labelled 43S complexes to fluorophores attached at defined positions on model mRNAs. This will enable us to determine whether scanning involves base-by-base inspection of a 5'NTR by the ribosome. Factor omission during initiation on a wholly unstructured 5'NTR will be used to differentiate between ATP hydrolysis during scanning by ribosomes and by associated eIF4A. The structural basis of interactions between eIFs 1 and 1A with other translation components (mRNA, 40S subunits and eIF3) will be characterized to elucidate the essential roles of these factors in scanning, initiation codon recognition and dissociating aberrant ribosomal complexes from mRNA.
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