THE SIGNAL FOR A LEAKY UAG STOP CODON IN SEVERAL PLANT-VIRUSES INCLUDES THE 2 DOWNSTREAM CODONS

Expression of the RNA replicase domain of tobacco mosaic virus (TMV) and certain protein-coding regions in other plant viruses, is mediated by translational readthrough of a leaky UAG stop codon. It has been proposed that normal tobacco tyrosine tRNAs are able to read the UAG codon of TMV by non-conventional base-pairing but recent findings that stop codons can also be bypassed as a result of extended translocational shifts (tRNA hopping) have encouraged a re-examination. In light of the alternatives, we investigated the sequences flanking the leaky UAG codon using an in vivo assay in which bypass of the stop codon is coupled to the transient expression of β-glucuronidase (GUS) reporter genes in tobacco protoplasts. Analysis of GUS constructions in which codons flanking the stop were altered allowed definition of the minimal sequence required for readthrough as UAG-CAA-UUA. The effects of all possible single-base mutations in the codons flanking the stop indicated that 3′ contexts of the form CAR-YYA confer leakiness and that the 3′ context permits readthrough of UAA and UGA stop codons as well as UAG. Our studies demonstrate a major role for the 3′ context in the readthrough process and do not support a model in which the UAG is bypassed exclusively as a result of anticodon-codon interactions. No evidence for tRNA hopping was obtained. The 3′ context apparently represents a unique sequence element that affects translation termination. © 1991.