SECONDARY STRUCTURE AND MUTATIONAL ANALYSIS OF THE RIBOSOMAL FRAMESHIFT SIGNAL OF ROUS-SARCOMA VIRUS

Expression of the Gag-Pol polyprotein of Rous sarcoma virus (RSV) requ ires a -1 ribosomal frameshifting event at the overlap region of the g ag and pol open reading frames. The signal for frameshifting is compos ed of two essential mRNA elements; a slippery sequence (AAAUUUA) where the ribosome changes reading frame, and a stimulatory RNA structure l ocated immediately downstream. This RNA is predicted to be a complex s tem-loop but may also form an RNA pseudoknot. We have investigated the structure of the RSV frameshift signal by a combination of enzymatic and chemical structure probing and site-directed mutagenesis. The stim ulatory RNA is indeed a complex stem-loop with a long stable stem and two additional stem-loops contained as substructures within the main l oop region. The substructures are not however required for frameshifti ng. Evidence for an additional interaction between a stretch of nucleo tides in the main loop and a region downstream to generate an RNA pseu doknot was obtained from an analysis-of the frameshifting properties o f RSV mutants translated in the rabbit reticulocyte lysate in vitro tr anslation system. Mutations that disrupted the predicted pseudoknot-fo rming sequences reduced frameshifting but when the mutations were comb ined and should re-form the pseudoknot, frameshifting was restored to a level approaching that of the wild-type construct. It was also obser ved that the predicted pseudoknot-forming regions had reduced sensitiv ity to cleavage by the single-stranded probe imidazole. Overall, howev er, the structure probing data indicate that the pseudoknot interactio n is weak and may form transiently. In comparison to other characteris ed RNA structures present at viral frameshift signals, the RSV stimula tor falls into a novel group. It cannot be considered to be a simple h airpin-loop yet it is distinct from other well characterised frameshif t-inducing RNA pseudoknots in that the overall contribution of the RSV pseudoknot to frameshifting is less dramatic. (C) 1998 Academic Press .