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ABSTRACT: Expression of the Gag-Pol polyprotein of Rous sarcoma virus (RSV) requires a -1 ribosomal frameshifting event at the overlap region of the gag and pol open reading frames. The signal for frameshifting is composed of two essential mRNA elements; a slippery sequence (AAAUUUA) where the ribosome changes reading frame, and a stimulatory RNA structure located immediately downstream. This RNA is predicted to be a complex stem-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 stimulatory RNA is indeed a complex stem-loop with a long stable stem and two additional stem-loops contained as substructures within the main loop region. The substructures are not however required for frameshifting. Evidence for an additional interaction between a stretch of nucleotides in the main loop and a region downstream to generate an RNA pseudoknot was obtained from an analysis of the frameshifting properties of RSV mutants translated in the rabbit reticulocyte lysate in vitro translation system. Mutations that disrupted the predicted pseudoknot-forming sequences reduced frameshifting but when the mutations were combined and should re-form the pseudoknot, frameshifting was restored to a level approaching that of the wild-type construct. It was also observed that the predicted pseudoknot-forming regions had reduced sensitivity to cleavage by the single-stranded probe imidazole. Overall, however, the structure probing data indicate that the pseudoknot interaction is weak and may form transiently. In comparison to other characterised RNA structures present at viral frameshift signals, the RSV stimulator falls into a novel group. It cannot be considered to be a simple hairpin-loop yet it is distinct from other well characterised frameshift-inducing RNA pseudoknots in that the overall contribution of the RSV pseudoknot to frameshifting is less dramatic.
Journal of Molecular Biology 12/1998; 284(2):205-25. · 4.00 Impact Factor
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ABSTRACT: Eukaryotic ribosomal frameshift signals generally contain two elements, a heptanucleotide slippery sequence (XXXYYYN) and an RNA secondary structure, often an RNA pseudoknot, located downstream. Frameshifting takes place at the slippery sequence by simultaneous slippage of two ribosome-bound tRNAs. All of the tRNAs that are predicted to decode frameshift sites in the ribosomal A-site (XXXYYYN) possess a hypermodified base in the anticodon-loop and it is conceivable that these modifications play a role in the frameshift process. To test this, we expressed slippery sequence variants of the coronavirus IBV frameshift signal in strains of Escherichia coli unable to modify fully either tRNA(Lys) or tRNA(Asn). At the slippery sequences UUUAAAC and UUUAAAU (underlined codon decoded by tRNA(Asn), anticodon 5' QUU 3'), frameshifting was very inefficient (2 to 3%) and in strains deficient in the biosynthesis of Q base, was increased (AAU) or decreased (AAC) only two-fold. In E. coli, therefore, hypomodification of tRNA(Asn) had little effect on frameshifting. The situation with the efficient slippery sequences UUUAAAA (15%) and UUUAAAG (40%) (underlined codon decoded by tRNA(Lys), anticodon 5' mnm5s2UUU 3') was more complex, since the wobble base of tRNA(Lys) is modified at two positions. Of four available mutants, only trmE (s2UUU) had a marked influence on frameshifting, increasing the efficiency of the process at the slippery sequence UUUAAAA. No effect on frameshifting was seen in trmC1 (cmnm5s2UUU) or trmC2 (nm5s2UUU) strains and only a very small reduction (at UUUAAAG) was observed in an asuE (mnm5UUU) strain. The slipperiness of tRNA(Lys), therefore, cannot be ascribed to a single modification site on the base. However, the data support a role for the amino group of the mnm5 substitution in shaping the anticodon structure. Whether these conclusions can be extended to eukaryotic translation systems is uncertain. Although E. coli ribosomes changed frame at the IBV signal (UUUAAAG) with an efficiency similar to that measured in reticulocyte lysates (40%), there were important qualitative differences. Frameshifting of prokaryotic ribosomes was pseudoknot-independent (although secondary structure dependent) and appeared to require slippage of only a single tRNA.
Journal of Molecular Biology 08/1997; 270(3):360-73. · 4.00 Impact Factor
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ABSTRACT: The genomic RNA of human astrovirus serotype 1 (HAst-1) contains three open reading frames (ORFs), 1a, 1b, and 2. ORF 1b is located downstream of, and overlaps, 1a, and it has been suggested on the basis of sequence analysis that expression of ORF 1b is mediated through -1 ribosomal frameshifting. To examine this possibility, a cDNA fragment containing the 1a-1b overlap region was cloned within a reporter gene and placed under the control of the bacteriophage SP6 promoter in a recombinant plasmid. Synthetic transcripts derived from this plasmid, when translated in the rabbit reticulocyte lysate cell-free system, specified the synthesis of polypeptides whose size and antibody reactivity were consistent with an efficient -1 ribosomal frameshift event at the overlap region. The HAst-1 frameshift signal has two essential components, a heptanucleotide slippery sequence, A6C, and a stem-loop structure in the RNA. The presence of this structure was confirmed by complementary and compensatory mutation analysis and by direct structure probing with single- and double-stranded RNA-specific reagents. The HAst-1 frameshift signal, like that present at the overlap of the gag and pro genes of the retrovirus human T-cell lymphotrophic virus type II, does not involve the formation of an RNA pseudoknot.
Journal of Virology 10/1994; 68(9):5588-95. · 5.40 Impact Factor
