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 beta-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 read through 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 read 
through of UAA and UGA stop codons as well as UAG. Our studies demonstrate a 
major role for the 3' context in the read through process and do not support a 
model in which teh 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.