In vitro trans translation mediated by alanine-charged 10Sa RNA

10Sa RNA is a bacterial small stable RNA, in which the 5' and 3'-terminal sequences can be folded into a tRNA-like secondary structure which can be aminoacylated with alanine. It was found that Escherichia coli 10Sa RNA facilitated the incorporation of alanine, tyrosine, aspartic acid and glutamic acid, but not valine, isoleucine, serine or arginine, into the growing polypeptide in vitro, depending on poly (U)-directed poly phenylalanine synthesis. This result indicates that 10Sa RNA functions as an mRNA for the tag-peptide which has been found to be attached to the C termini of truncated polypeptides synthesized in vitro. Aminoacylation with alanine was required for tag-specific amino acid incorporation and for efficient association of 10Sa RNA with the ribosome, indicating that 10Sa RNA also functions as an alanine tRNA in the tag-peptide synthesis. The dual function of 10Sa RNA both as an mRNA and as a tRNA in vitro strongly supports the trans translation hypothesis. (C) 1997 Academic Press Limited.