Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus.

Abstract

Expression of the murine leukemia virus poi gene occurs by translational readthrough of an in-frame UAG codon between the gag and poi coding regions. In a previous study, we mutated the UAG codon to UAA or UGA and demonstrated that both of these termination codons could be suppressed in reticulocyte lysates and in infected cells with the same efficiency as UAG. We now report the identity of the amino acids inserted in vitro in response to UAA and UGA in fusion products containing the gag-pol junction region. The results show that UAA, like UAG, directs the incorporation of glutamine, whereas UGA directs the incorporation of three amino acids, arginine, cysteine, and tryptophan. To our knowledge, this is the first report indicating misreading of UAA as glutamine and UGA as arginine and cysteine in higher eukaryotes. Interestingly, although our protein synthesis system presumably contains other known UAG and UGA suppressors, these tRNAs did not suppress the termination codons in our experiments. Thus, it seems possible that the sequence surrounding the gag-pol junction not only promotes suppression but also helps determine which tRNAs function in suppression.Readthrough suppression of termination codons is a mechanism of translational regulation that is used by a number of viruses infecting higher eukaryotes. The analysis of this phenomenon thus provides new information on the functional capabilities of the translational apparatus of these organisms. We have been investigating suppression in the murine leukemia virus (MuLV) system (1, 2). MuLV, like other mammalian type C retroviruses, regulates pol gene expression by translational readthrough ofan in-frame UAG codon between the gag and pol coding regions (3-5). Suppression of this termination codon occurs with a frequency of about 5% (3)(4)(5) and is mediated by insertion of a glutamine residue at the position corresponding to the UAG (6). The readthrough product is a 200-kDa gag-pol fusion protein, which is cleaved at a late stage in virus assembly to give rise to protease, reverse transcriptase, and integrase (3).To determine whether the signals that govern readthrough are unique for UAG, we used oligonucleotide-directed mutagenesis to change the UAG at the Moloney (Mo)-MuLV gag-pol junction to UAA or UGA (2). Interestingly, we found that these termination codons could be efficiently suppressed both in reticulocyte lysates and in infected cells (2). These results demonstrated that the suppression signals are not specific for UAG and that mammalian cells and cell extracts contain tRNAs capable of suppressing UGA and UAA codons which appear in a retroviral context. Similar findings have been reported by other investigators working with tobacco mosaic virus (TMV) (7), Sindbis virus (8), and MuLV (9).In the present study we have identified the amino acids inserted in response to UAA and UGA by using an in vitro MuLV suppression system (1). We find that UAA, like UAG, directs the incorporation of glutamine into protein, whereas UGA directs the incorporation ...

Complete nucleotide sequence of Colorado tick fever virus segments M6, S1 and S2.

“…The corresponding putative protein would consist of 602 amino acids. Similar situations have been reported in retroviruses (Feng et al, 1990) and alphaviruses (Strauss & Strauss, 1994). Such opal codons have been shown to be leaky, allowing a readthrough phenomenon resulting from the incorporation of arginine, cysteine or tryptophan in place of the opal codon (Feng et al, 1990).…”

“…Similar situations have been reported in retroviruses (Feng et al, 1990) and alphaviruses (Strauss & Strauss, 1994). Such opal codons have been shown to be leaky, allowing a readthrough phenomenon resulting from the incorporation of arginine, cysteine or tryptophan in place of the opal codon (Feng et al, 1990). The suppression of this codon was found to be directly dependent on the presence of the flanking cytosine (Strauss & Strauss, 1994).…”

The Amber Codon in the Gene Encoding the Monomethylamine Methyltransferase Isolated from Methanosarcina barkeri Is Translated as a Sense Codon

“…Of course, a naturally occurring stop codon can also be suppressed with a tRNA whose anticodon can base pair with the stop codon. Some eucaryotic tRNA species that suppress naturally occurring stop codons also decode normal sense codons (19,20). In contrast, dedicated tRNA species are involved in decoding UGA as selenocysteine in all three domains (21).…”

Evidence of abundant stop codon readthrough in Drosophila and other metazoa

“…Although no cognate stop suppressor tRNA genes have been found in the Drosophila genome (Chan and Lowe 2009), experiments in Drosophila, mammals, and yeast have shown that specific near-cognate tRNAs can insert an amino acid when a stop codon is read through, with glutamine or tyrosine incorporated for UAA, glutamine, tyrosine, leucine, lysine, or possibly low levels of tryptophan for UAG, and arginine, cysteine, serine, or tryptophan for UGA (Bienz and Kubli 1981;Pure et al 1985;Valle et al 1987;Feng et al 1990;Fearon et al 1994;Chittum et al 1998;Lao et al 2009;Supplemental Text S5). Thus, the possible amino acids inserted for UGA are mostly different from the ones inserted for UAA or UAG, while UAA and UAG are more likely to result in the same amino acid incorporation.…”