EVIDENCE FOR INVOLVEMENT OF TRANS-ACTING FACTORS IN SELECTION OF THE AUG START CODON DURING EUKARYOTIC TRANSLATIONAL INITIATION

The molecular mechanism with which an appropriate AUG codon is selecte d as the start site for translational initiation by eukaryotic ribosom es is not known. By using a cell-free translation system, small RNA mo lecules containing single AUG codons, surrounded by various nucleotide sequences, were tested for their abilities to interfere,vith the tran slation of a reporter mRNA. RNAs containing the AUG in an ACCAUGG cont ext (Kozak consensus sequence) were able to inhibit translation of the reporter mRNA. In contrast, RNAs containing the AUG in a less favorab le context for start site selection (for example, CAGAUGC) had no effe ct on the translation of the reporter mRNA. The effect mediated by the ACCAUGG-containing RNAs was not due to sequestration of ribosomal sub units or to particular structural features in these RNAs. To identify potential trans-acting factors that might be preferentially bound by A CCAUGG-containing RNAs, ACCAUGG- and CAGAUGC-containing RNAs with a si ngle 4-thiouridine residue at the AUG were intubated with partially fr actionated extracts, and AUG-binding proteins were identified after ir radiation of the complexes with UV light and subsequent analysis by ge l electrophoresis. The analysis of such complexes in competition exper iments revealed that proteins, approximately 50 and 100 kDa in size, w ere found to bind directly at the AUG codon embedded in the ACCAUGG mo tif. One of these proteins has been identified as the La autoantigen. These findings indicate that trans-acting factors may play a role in A UG start site selection during translational initiation.