Inhibition of Translation Termination Mediated by an Interaction of Eukaryotic Release Factor 1 with a Nascent Peptidyl-tRNA

Abstract

Expression of the human cytomegalovirus UL4 gene is inhibited by translation of a 22-codon-upstream open reading frame (uORF2). The peptide product of uORF2 acts in a sequence-dependent manner to inhibit its own translation termination, resulting in persistence of the uORF2 peptidyl-tRNA linkage. Consequently, ribosomes stall at the uORF2 termination codon and obstruct downstream translation. Since termination appears to be the critical step affected by translation of uORF2, we examined the role of eukaryotic release factors 1 and 3 (eRF1 and eRF3) in the inhibitory mechanism. In support of the hypothesis that an interaction between eRF1 and uORF2 contributes to uORF2 inhibitory activity, specific residues in each protein, glycines 183 and 184 of the eRF1 GGQ motif and prolines 21 and 22 of the uORF2 peptide, were found to be necessary for full inhibition of downstream translation. Immunoblot analyses revealed that eRF1, but not eRF3, accumulated in the uORF2-stalled ribosome complex. Finally, increased puromycin sensitivity was observed after depletion of eRF1 from the stalled ribosome complex, consistent with inhibition of peptidyl-tRNA hydrolysis resulting from an eRF1-uORF2 peptidyl-tRNA interaction. These results reveal the paradoxical potential for interactions between a nascent peptide and eRF1 to obstruct the translation termination cascade.Expression of the human cytomegalovirus UL4 gene is controlled by an unusual translational mechanism in which termination is the critical regulatory step (1,21,28,36). The UL4 coding region is present on a transcript that contains three upstream open reading frames (uORFs), the second of which (uORF2) encodes a 22-amino-acid peptide that inhibits downstream translation in cis. By a mechanism that depends on the sequence of the encoded peptide, uORF2 inhibits termination at its own stop codon, resulting in a persistent covalent linkage between the nascent uORF2 peptide and the terminal cognate tRNA Pro . The termination arrest causes translating ribosomes to stall over the uORF2 stop codon both in cell-free translation assays and in cells, setting up a blockade that prevents scanning ribosomes from leaking past uORF2 to gain access to the UL4 start codon.Translation termination is a complex process that involves stop codon recognition, peptidyl-tRNA hydrolysis, and release of ribosome from the message (29). While the termination process is incompletely characterized mechanistically, the participation of protein factors has been elucidated. The class I eukaryotic release factor (eRF1) is responsible for stop codon recognition, ribosome binding, and triggering hydrolysis of the peptidyl-tRNA bond (14). Meanwhile, eRF3 stimulates eRF1 activity in a GTP-dependent fashion (50). In the uORF2 system, the persistent linkage of the nascent uORF2 peptide to tRNA Pro indicates that translation of uORF2 interferes with or prior to the peptidyl-tRNA hydrolysis step in the termination cascade. Since translational inhibition depends on the presence of a stop codon at the end of uORF2...

The canonical UPF1-dependent nonsense-mediated mRNA decay is inhibited in transcripts carrying a short open reading frame independent of sequence context

“…The percentage mRNA values relative to normal were plotted for each construct, and standard deviations from three independent experiments are shown. not limited to releasing factors, can inhibit translation elongation as well as termination (Morris and Geballe 2000;Janzen et al 2002). In the case of NMD, UPF1 must interact with eRF1 and eRF3 to terminate translation and, in the proper setting, to interact with additional factors to trigger NMD (Gaba et al 2005;Kashima et al 2006).…”

uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

“…When present as the last or penultimate amino acid, proline has major effects on translation termination, and this feature can be exploited. [An example with tandem prolines is in the human cytomegalovirus UL4 uORF (17). An example with a single proline is in the decoding of the Escherichia coli tryptophanase operon (18).…”

Cellular mRNAs access second ORFs using a novel amino acid sequence-dependent coupled translation termination–reinitiation mechanism

“…The reasons for this are not yet clear but it may be due to stalled termination of translation of ORF-1 inhibiting reinitiation. This may be similar to the situation described for the cytomegalovirus UL4 gene in which a delay in cleavage of the final aminoacyl tRNA peptidyl bond in a protein encoded by a short ORF upstream of the main ORF results in stalling of termination and subsequent reduction in translation of the primary product (Degnin et al 1993;Janzen et al 2002). In the situation of an overlapping ORF-1 and ORF-2 this may also lead to physical occlusion of the ORF-2 initiation codon to ribosomes that would generate the result seen.…”