On the mechanism of delayed inhibition of protein synthesis in heme-defecient rabbit reticulocyte lysates. (1/30)

In the absence of added hemin, protein synthesis in a rabbit reticulocyte lysate declines abruptly (shuts off) after about 5 min at 30 degrees. In these studies we have examined the basis for the lag period preceding shut-off. The initiation factor that binds Met-tRNAf, previously shown to be rate-limiting in inhibited, heme-deficient lysates, is found to be used stoichiometrically in the presence of excess inhibitor. We suggest that a principal effect of the inhibitor is to impair the recycling of the Met-tRNAf-binding factor; the lag period is attributable largely to the presence of a pool of excess Met-tRNAf-binding factor, which, once used in initiation, cannot be recycled because of the action of the inhibitor.  (+info)

The binding sites for tRNA on eukaryotic ribosomes. (2/30)

We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes.  (+info)

Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. (3/30)

The small ribosomal subunit is responsible for the decoding of genetic information and plays a key role in the initiation of protein synthesis. We analyzed by X-ray crystallography the structures of three different complexes of the small ribosomal subunit of Thermus thermophilus with the A-site inhibitor tetracycline, the universal initiation inhibitor edeine and the C-terminal domain of the translation initiation factor IF3. The crystal structure analysis of the complex with tetracycline revealed the functionally important site responsible for the blockage of the A-site. Five additional tetracycline sites resolve most of the controversial biochemical data on the location of tetracycline. The interaction of edeine with the small subunit indicates its role in inhibiting initiation and shows its involvement with P-site tRNA. The location of the C-terminal domain of IF3, at the solvent side of the platform, sheds light on the formation of the initiation complex, and implies that the anti-association activity of IF3 is due to its influence on the conformational dynamics of the small ribosomal subunit.  (+info)

Internal initiation in Saccharomyces cerevisiae mediated by an initiator tRNA/eIF2-independent internal ribosome entry site element. (4/30)

Internal initiation of translation can be mediated by specific internal ribosome entry site (IRES) elements that are located in certain mammalian and viral mRNA molecules. Thus far, these mammalian cellular and viral IRES elements have not been shown to function in the yeast Saccharomyces cerevisiae. We report here that a recently discovered IRES located in the genome of cricket paralysis virus can direct the efficient translation of a second URA3 cistron in dicistronic mRNAs in S. cerevisiae, thereby conferring uracil-independent growth. Curiously, the IRES functions poorly in wild-type yeast but functions efficiently either in the presence of constitutive expression of the eIF2 kinase GCN2 or in cells that have two initiator tRNA(met) genes disrupted. Both of these conditions have been shown to lower the amounts of ternary eIF2-GTP/initiator tRNA(met) complexes. Furthermore, tRNA(met)-independent initiation was also observed in translation-competent extracts prepared from S. cerevisiae in the presence of edeine, a compound that has been shown to interfere with start codon recognition by ribosomal subunits carrying ternary complexes. Therefore, the cricket paralysis virus IRES is likely to recruit ribosomes by internal initiation in S. cerevisiae in the absence of eIF2 and initiator tRNA(met), by the same mechanism of factor-independent ribosome recruitment used in mammalian cells. These findings will allow the use of yeast genetics to determine the mechanism of internal ribosome entry.  (+info)

Dissecting the ribosomal inhibition mechanisms of edeine and pactamycin: the universally conserved residues G693 and C795 regulate P-site RNA binding. (5/30)

The crystal structures of the universal translation-initiation inhibitors edeine and pactamycin bound to ribosomal 30S subunit have revealed that edeine induces base pairing of G693:C795, residues that constitute the pactamycin binding site. Here, we show that base pair formation by addition of edeine inhibits tRNA binding to the P site by preventing codon-anticodon interaction and that addition of pactamycin, which rebreaks the base pair, can relieve this inhibition. In addition, edeine induces translational misreading in the A site, at levels comparable to those induced by the classic misreading antibiotic streptomycin. Binding of pactamycin between residues G693 and C795 strongly inhibits translocation with a surprising tRNA specificity but has no effect on translation initiation, suggesting that reclassification of this antibiotic is necessary. Collectively, these results suggest that the universally conserved G693:C795 residues regulate tRNA binding at the P site of the ribosome and influence translocation efficiency.  (+info)

In vitro transcription of Tomato spotted wilt virus is independent of translation. (6/30)

Ongoing transcription in vitro of Tomato spotted wilt virus (TSWV) has previously been demonstrated to require the presence of reticulocyte lysate. This dependence was further investigated by testing the occurrence of transcription in the presence of two translation inhibitors: edeine, an inhibitor that still allows scanning of nascent mRNAs by the 40S ribosomal subunit, and cycloheximide, an inhibitor that completely blocks translation including ribosome scanning. Neither of these inhibitors blocked TSWV transcription initiation or elongation in vitro, as demonstrated by de novo-synthesized viral mRNAs with globin mRNA-derived leader sequences, suggesting that TSWV transcription in vitro requires the presence of (a component within) reticulocyte lysate, rather than a viral protein resulting from translation.  (+info)

Factor-independent assembly of elongation-competent ribosomes by an internal ribosome entry site located in an RNA virus that infects penaeid shrimp. (7/30)

The Taura syndrome virus (TSV), a member of the Dicistroviridae family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNAmet. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.  (+info)

Translation of the F protein of hepatitis C virus is initiated at a non-AUG codon in a +1 reading frame relative to the polyprotein. (8/30)

The hepatitis C virus (HCV) genome contains an internal ribosome entry site (IRES) followed by a large open reading frame coding for a polyprotein that is cleaved into 10 proteins. An additional HCV protein, the F protein, was recently suggested to result from a +1 frameshift by a minority of ribosomes that initiated translation at the HCV AUG initiator codon of the polyprotein. In the present study, we reassessed the mechanism accounting for the synthesis of the F protein by measuring the expression in cultured cells of a luciferase reporter gene with an insertion encompassing the IRES plus the beginning of the HCV-coding region preceding the luciferase-coding sequence. The insertion was such that luciferase expression was either in the +1 reading frame relative to the HCV AUG initiator codon, mimicking the expression of the F protein, or in-frame with this AUG, mimicking the expression of the polyprotein. Introduction of a stop codon at various positions in-frame with the AUG initiator codon and substitution of this AUG with UAC inhibited luciferase expression in the 0 reading frame but not in the +1 reading frame, ruling out that the synthesis of the F protein results from a +1 frameshift. Introduction of a stop codon at various positions in the +1 reading frame identified the codon overlapping codon 26 of the polyprotein in the +1 reading frame as the translation start site for the F protein. This codon 26(+1) is either GUG or GCG in the viral variants. Expression of the F protein strongly increased when codon 26(+1) was replaced with AUG, or when its context was mutated into an optimal Kozak context, but was severely decreased in the presence of low concentrations of edeine. These observations are consistent with a Met-tRNA(i)-dependent initiation of translation at a non-AUG codon for the synthesis of the F protein.  (+info)

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