Inhibition of translation termination mediated by an interaction of eukaryotic release factor 1 with a nascent peptidyl-tRNA

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.     

Translational Effects of Mutations and Polymorphisms in a Repressive Upstream Open Reading Frame of the Human Cytomegalovirus UL4 Gene

The human cytomegalovirus (HCMV) gpUL4 mRNA contains a 22-codon upstream open reading frame (uORF2), the peptide product of which represses downstream translation by blocking translation termination at its own stop codon and by causing ribosomes to stall on the mRNA. A distinctive feature of this unusual mechanism is its strict dependence on the uORF2 peptide sequence. To delineate sequence elements that function in the inhibitory mechanism, deletions and missense mutations affecting the previously uncharacterized amino-terminal region of uORF2 were analyzed in transient-transfection and infection assays. These experiments identified multiple codons in this region that are necessary for inhibition of downstream translation by uORF2 and, in conjunction with previous results, demonstrated that amino acids dispersed throughout the uORF2 peptide participate in the repressive mechanism. In contrast to the highly conserved carboxy terminus, the amino-terminal portion of the uORF2 peptide is polymorphic. A survey of uORF2 sequences in HCMV clinical isolates revealed that although most have uORF2 sequences that are predicted to retain the uORF2 inhibitory activity, approximately 15% contain polymorphisms at codons that are essential for full inhibition by uORF2. Consistent with predictions based on analyses of engineered mutations, two viral isolates with uORF2 sequences that do not inhibit downstream translation in transfection assays expressed much more gpUL4 protein but similar levels of UL4 mRNA compared to the levels produced by the prototypic laboratory strain HCMV (Towne) and another clinical isolate with an inhibitory variant uORF2. These results demonstrate that uORF2 is polymorphic in sequence and repressive activity and suggest that the uORF2 regulatory mechanism, although prevalent among natural HCMV isolates, is not absolutely essential for viral replication.     

Physical evidence for distinct mechanisms of translational control by upstream open reading frames.

The Saccharomyces cerevisiae GCN4 mRNA 5'-leader contains four upstream open reading frames (uORFs) and the CPA1 leader contains a single uORF. To determine how these uORFs control translation, we examined mRNAs containing these leaders in cell-free translation extracts to determine where ribosomes were loaded first and where they were loaded during steady-state translation. Ribosomes predominantly loaded first at GCN4 uORF1. Following its translation, but not the translation of uORF4, they efficiently reinitiated protein synthesis at Gcn4p. Adding purified eIF2 increased reinitiation at uORFs 3 or 4 and reduced reinitiation at Gcn4p. This indicates that eIF2 affects the site of reinitiation following translation of GCN4 uORF1 in vitro. In contrast, for mRNA containing the CPA1 uORF, ribosomes reached the downstream start codon by scanning past the uORF. Addition of arginine caused ribosomes that had synthesized the uORF polypeptide to stall at its termination codon, reducing loading at the downstream start codon, apparently by blocking scanning ribosomes, and not by affecting reinitiation. The GCN4 and CPA1 uORFs thus control translation in fundamentally different ways.     

Ribosome stalling is responsible for arginine-specific translational attenuation in Neurospora crassa.

The Neurospora crassa arg-2 upstream open reading frame (uORF) plays a role in negative arginine-specific translational regulation. Primer extension inhibition analyses of arg-2 uORF-containing RNA translated in a cell-free system in which arginine-specific regulation was retained revealed "toeprints" corresponding to ribosomes positioned at the uORF initiation and termination codons and at the downstream initiation codon. At high arginine concentrations, the toeprint signal corresponding to ribosomes at the uORF termination codon rapidly increased; a new, broad toeprint that represents additional ribosomes stalled on the uORF appeared 21 to 30 nucleotides upstream of this site; and the toeprint signal corresponding to ribosomes at the downstream initiation codon decreased. These data suggest that arginine increases ribosomal stalling and thereby decreases translation from the downstream initiation codon.     

In vitro translation of the upstream open reading frame in the mammalian mRNA encoding S-adenosylmethionine decarboxylase

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive element that suppresses translation of the associated downstream cistron in vivo. In this paper, we provide the first direct evidence of peptide synthesis from the S-adenosylmethionine decarboxylase uORF using an in vitro translation system. We examine both the influence of cation concentration on peptide synthesis and the effect of altering the uORF sequence on peptide synthesis. Synthesis of wild type and altered peptides was similar at all concentrations of magnesium tested. In contrast, synthesis of the wild type peptide was more sensitive than that of altered peptides to elevated concentrations of the naturally occurring polyamines, spermidine and spermine, as well as several polyamine analogs. The sensitivity of in vitro synthesis to spermidine was influenced by both the amino acid sequence and the length of the peptide product of the uORF. Findings from the present study correlate with the effects of the uORF and polyamines on translation of a downstream cistron in vivo and support the hypothesis that polyamines and the structure of the nascent peptide create a rate-limiting step in uORF translation, perhaps through a ribosome stalling mechanism.     

Polyamine regulation of ribosome pausing at the upstream open reading frame of S-adenosylmethionine decarboxylase

Synthesis of S-adenosylmethionine decarboxylase (AdoMetDC), a key regulated enzyme in the pathway of polyamine biosynthesis, is feedback-controlled at the level of translation by spermidine and spermine. The peptide product of an upstream open reading frame (uORF) in the mRNA is solely responsible for polyamine regulation of AdoMetDC translation. Using a primer extension inhibition assay and in vitro protein synthesis reactions, we found ribosomes paused at or close to the termination codon of the uORF. This pause was greatly diminished with the altered uORFs' sequences that abolish uORF regulation in vivo. The half-life of the ribosome pause was related to the concentration of polyamines present but was unaffected by magnesium concentration. Furthermore, inhibition of translation initiation at a reporter gene placed downstream of the AdoMetDC uORF directly correlated with the stability of the ribosome pause at the uORF. These observations are consistent with a model in which regulation of ribosome pausing at the uORF by polyamines controls ribosome access to the downstream AdoMetDC reading frame.     

Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders

We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.     

The Evolutionarily Conserved Eukaryotic Arginine Attenuator Peptide Regulates the Movement of Ribosomes That Have Translated It

Translation of the upstream open reading frame (uORF) in the 5′ leader segment of the Neurospora crassa arg-2 mRNA causes reduced initiation at a downstream start codon when arginine is plentiful. Previous examination of this translational attenuation mechanism using a primer-extension inhibition (toeprint) assay in a homologous N. crassa cell-free translation system showed that arginine causes ribosomes to stall at the uORF termination codon. This stalling apparently regulates translation by preventing trailing scanning ribosomes from reaching the downstream start codon. Here we provide evidence that neither the distance between the uORF stop codon and the downstream initiation codon nor the nature of the stop codon used to terminate translation of the uORF-encoded arginine attenuator peptide (AAP) is important for regulation. Furthermore, translation of the AAP coding region regulates synthesis of the firefly luciferase polypeptide when it is fused directly at the N terminus of that polypeptide. In this case, the elongating ribosome stalls in response to Arg soon after it translates the AAP coding region. Regulation by this eukaryotic leader peptide thus appears to be exerted through a novel mechanism of cis-acting translational control.     

A quantitative model for translational control of the GCN4 gene of Saccharomyces cerevisiae

Expression of the GCN4 gene of Saccharomyces cerevisiae is regulated at the translational level by short open reading frames (uORFs) present in the leader sequence of its mRNA. Under conditions of amino acid sufficiency, these sequences restrict the flow of initiating ribosomes to the GCN4 AUG start codon. Mutational analysis of GCN4 has led to a model in which ribosomes must translate the 5'-proximal uORF1 and reassemble an initiation complex in order to translate GCN4. This reassembly process is thought to be rapid when amino acids are abundant, such that reinitiation occurs at uORF2, uORF3, or uORF4. Reinitiation at these sites prevents translation of GCN4, presumably because ribosomes dissociate from the mRNA following termination at uORFs 2 to 4. Because of reduced initiation factor activity under starvation conditions, a substantial fraction of ribosomal subunits scanning downstream from uORF1 are not ready to reinitiate when they reach uORFs 2 to 4, but become competent to do so while scanning the additional sequences between uORF4 and GCN4. Examination of the effects of point mutations in the ATG codons of the different uORFs suggests a quantitative model for this control mechanism that describes the probability of reinitiation as a function of the distance scanned downstream from uORF1. This model accounts for the phenotypes of a number of deletion and insertion mutations that alter the intercistronic spacing between the uORFs and GCN4. The correspondence between observed and predicted results implies that the differential rates of reinitiation at GCN4 versus uORFs 2 to 4 are determined largely by the different scanning times required to reach each of these start sites following translation of uORF1. In addition, it supports the notion that an increased scanning-time requirement for reinitiation in amino acid-starved cells forms the basis for translational derepression of GCN4 expression.