Initiation Factor Requirements for the in vitro Synthesis of T₄ Lysozyme

Abstract

PROTEIN synthesis requires initiation of translation at the correct site on mRNA. Two initiation factors, A and C (ref. 1), or F1 and F2 (ref. 2), promote the ApUpG-dependent attachment of formylmethionyl-tRNA (fmet-tRNA_f) to Escherichia coli ribosomes^2–4, and account for initiation AUG triplets^5,6. A mechanism must exist, however, for the discrimination between internal and initial methionine codons. In order to study the possible role of the third initiation factor, B, in this selection mechanism, we have examined the role of the three factors in the synthesis of lysozyme promoted by T₄ mRNA⁷. We assumed that polypeptide synthesis as measured by amino-acid incorporation should not necessarily require correct initiation, while active enzyme formation should be highly dependent on this process. We have used the ratio of enzyme formed to valine incorporated as a measure of the proportion of polypeptide molecules correctly initiated.