Translation of poliovirus RNA in vitro: Studies on n-formylmethionine-labeled polypeptides initiated in cell-free extracts prepared from poliovirus infected HeLa cells

Abstract

Previous studies in our laboratory have provided evidence that cell-free systems translating the Mahoney strain of poliovirus type I RNA utilize two unique initiation sites. This study shows it is possible to resolve two major n-formyl[ 35S]methionin [ 35S]fmet) polypeptides by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the relative proportions of which show a Mg 2+ concentration dependence which correlates with that shown by the two previously identified amino terminal tryptic peptides. Tryptic peptide analysis of fmet-labeled polypeptides recovered from SDS-gel confirms this correlation, showing that tryptic peptide II is the amino terminal tryptic peptide of a high molecular polypeptide (115,000 daltons) and that tryptic peptide I is the amino terminus of a low molecular weight polypeptide (5–10,000 daltons). In this study ribosomal salt wash was added to the reaction mixtures to stimulate incorporation. We find that ribosomal salt wash prepared from mock-infected HeLa cells stimulates the in vitro translation of both polypeptides while salt wash prepared from poliovirus infected cells preferentially stimulates the utilization of the initiation site defined by tryptic peptide I.