Plant RNA viruses commonly exploit leaky translation termination signals in 
order to express internal protein coding regions. As a first step to elucidate 
the mechanism(s) by which ribosomes bypass leaky stop codons in vivo, we have 
devised a system in which readthrough is coupled to the transient expression of 
beta-glucuronidase (GUS) in tobacco protoplasts. GUS vectors that contain the 
stop codons and surrounding nucleotides from the readthrough regions of several 
different RNA viruses were constructed and the plasmids were tested for the 
ability to direct transient GUS expression. These studies indicated that 
ribosomes bypass the leaky termination sites at efficiencies ranging from 
essentially 0 to ca. 5% depending upon the viral sequence. The results suggest 
that the efficiency of readthrough is determined by the sequence surrounding the 
stop codon. We describe improved GUS expression vectors and optimized 
transfection conditions which made it possible to assay low-level translational 
events.