Identification and analysis of the pseudoknot-containing gag-pro ribosomal frameshift signal of simian retrovirus-1.

Abstract

The pro and pol genes of simian retrovirus-1 (SRV-1) are expressed as parts of a fusion protein generated by -1 ribosomal frameshifting. To investigate the requirements for frameshifting at the gag-pro overlap, we have inserted a stretch of 58 nucleotides containing the proposed frameshift signal into a plasmid that allows monitoring of translation in all three reading frames. In vitro translation of mRNAs derived from this plasmid indicated that the 58 nucleotides from the SRV-1 gag-pro overlap were sufficient to induce an efficient -1 shift in a heterologous context. Mutational analysis demonstrated that the slip site is formed at the heptanucleotide G GGA AAC. The frameshift efficiency of the wild type sequence in rabbit reticulocyte lysate was 23%. A second component of the frameshift signal is formed by a pseudoknot seven bases downstream of the slip site. The presence of this pseudoknot was confirmed by mutational analysis, employing complementary and compensatory base changes, and by probing the structure of short RNA transcripts containing the frameshift signal. Adding increasing amounts of an SRV-1 pseudoknot containing RNA transcript to a translation reaction programmed with an SRV-1 frameshift reporter mRNA had no effect on the frameshift efficiency, arguing against the role of a specific pseudoknot-recognising factor in the frameshifting process.