Inhibition of nascent-peptide release at translation termination

Abstract

The transcript leader of the human cytomegalovirus (CMV) gpUL4 (gp48) gene contains a 22-codon upstream open reading frame (uORF2) that represses translation of the downstream cistron. Previous work demonstrated that ribosomes stall at the termination codon of uORF2 and, remarkably, that the coding information of uORF2 is required for both the translational repression and ribosomal stalling. We now provide evidence that the peptide product of uORF2 is synthesized and is retained in the ribosome in the form of a peptidyl-tRNA. Translation of the gp48 transcript leader in cell extracts produces the 2.4-kDa uORF2 peptide and a second product migrating with an apparent molecular mass of 20 kDa that represents the uORF2 peptide covalently linked to tRNA(Pro), the tRNA predicted to decode the carboxy-terminal codon of uORF2. The uORF2 peptidyl-tRNA is only detected after translation of RNAs containing uORF2 sequences that also inhibit downstream translation and cause ribosomal stalling. These data support a model in which the nascent uORF2 peptide blocks translation termination prior to hydrolysis of the peptidyl-tRNA bond. This blockade results in ribosomal stalling on the transcript leader which in turn impedes the access of ribosomes to the downstream cistron. This system illustrates that translation termination may be a critical step controlling expression of some eukaryotic genes.