UGA suppression by tRNA(CmCA)(Trp) occurs in diverse virus RNAs due to a limited influence of the codon context

Abstract

We have recently identified chloroplast and cytoplasmic tRNA(CmCA)(Trp) as the first natural UGA suppressor tRNAs in plants. The interaction of these tRNAs with UGA involves a Cm:A mismatch at the first anticodon position. We show here that tRNA(CmCA)(Trp) is incapable of misreading UAA and UAG codons in vitro, implying that unconventional base pairs are not tolerated in the middle anticodon position. Furthermore, we demonstrate that the ability of tRNA(CmCA)(Trp) to promote UGA read-through depends on a quite simple codon context. Part of the sequence surrounding the leaky UGA stop codon in tobacco rattle virus RNA-1 was subcloned into a zein reporter gene and read-through efficiency was measured by translation of RNA transcripts in wheat germ extract. A number of mutations in the codons adjacent to the UGA were introduced by site-directed mutagenesis. It was found that single nucleotide exchanges at either side of the UGA had little effect on read-through efficiency. A pronounced influence on suppression by tRNA(CmCA)(Trp) was seen only if 2 or 3 nt at the 3'-side of the UGA codon had been simultaneously replaced. As a consequence of the flexible codon context accepted by tRNA(CmCA)(Trp), this tRNA is able to misread the UGA in a number of plant and animal viral RNAs that use translational read-through for expression of some of their genes.