Secondary structure and mutational analysis of the ribosomal frameshift signal of rous sarcoma virus 1 1Edited by D. E. Draper

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.

Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design

“…3A). These include the "slippery sequence" of seven nucleotides in the 3Ј region of pro, as well as the stem-loop structure in the 5Ј region of pol (15,20).…”

Efficient stimulation of site-specific ribosome frameshifting by antisense oligonucleotides

“…The MMTV and HIV sites both revealed ∼ 40% morpholino oligonucleotide-induced ribosome frameshifting, whereas, the complete HIV and MMTV frameshift cassettes with their natural stimulators have been reported to induce ribosome frameshifting to ∼ 10% (Grentzmann et al 1998;Dinman et al 2002;Dulude et al 2002) and 20% (Jacks et al 1987;Chamorro et al 1992), respectively. Similarly, the MOAB morpholino:RNA hybrid induces nearly 25% frameshifting on the AAAUUUA Rouse Sarcoma Virus gag-pol shift site, where natural levels of frameshifting are only about 5% (Jacks et al 1988;Marczinke et al 1998). Two shift sites, the Simian Retrovirus-1 (SRV-1) gag-pro G GGA AAC and IBV UUUAAAC frameshift sites revealed slightly lower morpholino oligonucleotide-induced frameshifting levels than those induced by the natural stimulators.…”

Poliovirus Internal Ribosome Entry Segment Structure Alterations That Specifically Affect Function in Neuronal Cells: Molecular Genetic Analysis

“…Protocols for structure probing experiments were based on those of Marczinke et al (27). Specifically, reaction mixtures contained 30,000 cpm of 5Ј-end-labeled RNA transcripts (corresponding to approximately 0.1 g) and 10 g of unlabeled yeast tRNA as a carrier.…”