Kinetics of ribosomal pausing during programmed-1 translational frameshifting

In the Saccharomyces cerevisiae double-stranded RNA virus, programmed -1 ri bosomal frameshifting is responsible for translation of the second open rea ding frame of the essential viral RNA. A typical slippery site and downstre am pseudoknot are necessary for this frameshifting event, and previous work has demonstrated that ribosomes pause over the slippery site. The translat ional intermediate associated with a ribosome paused at this position is de tected, and, using in vitro translation and quantitative heelprinting, the rates of synthesis, the ribosomal pause time, the proportion of ribosomes p aused at the slippery site, and the fraction of paused ribosomes that frame shift are estimated. About 10% of ribosomes pause at the slippery site in v itro, and some 60% of these continue in the -1 frame. Ribosomes that contin ue in the -1 frame pause about 10 times longer than it takes to complete a peptide bond in vitro. Altering the rate of translational initiation alters the rate of frameshifting in vivo. Our in vitro and in vivo experiments ca n best be interpreted to mean that there are three methods by which ribosom es pass the frameshift site, only one of which results in frameshifting.