Ribosomal frameshifting requires a pseudoknot in the Saccharomyces cerevisiae double-stranded RNA virus

Abstract

The large double-stranded RNA of the Saccharomyces cerevisiae (yeast) virus has two large overlapping open reading frames on the plus strand, one of which is translated via a -1 ribosomal frameshift. Sequences including the overlapping region, placed in novel contexts, can direct ribosomes to make a -1 frameshift in wheat germ extract, Escherichia coli and S. cerevisiae. This sequence includes a consensus slippery sequence, GGGUUUA, and has the potential to form a pseudoknot 3' to the putative frameshift site. Based on deletion analysis, a region of 71 nucleotides including the potential pseudoknot and the putative slippery sequence is sufficient for frameshifting. Site-directed mutagenesis demonstrates that the pseudoknot is essential for frameshifting.

Structure, stability and function of RNA pseudoknots involved in stimulating ribosomal frameshifting

“…In most instances where translational readthrough or ribosomal frameshifting is suspected or con®rmed to occur, the RNA sequence 5 to 9 nt downstream from the gag termination codon or the 3 H end of the slippery sequence is known or postulated to harbor an H-type pseudoknotted RNA structure (Figure 2(b)). The existence of these pseudoknots and their essential involvement in ribosomal frameshifting or read-through events have been con®rmed by mutagenesis studies in a number of viruses, including the coronavirus avian infectious bronchitis virus (IBV) (Brierley et al, 1989(Brierley et al, , 1991; animal retroviruses including RSV (Jacks et al, 1988a,b), mouse mammary tumor virus (MMTV) , feline immunode®ciency virus (FIV) (Morikawa & Bishop, 1992), simian retrovirus type-1 (SRV-1) (ten Dam et al, 1994), and MuLV (Wills et al, 1991;Feng et al, 1992); double-stranded RNA viruses of Saccharomyces cerevisiae, L-A and L-1 (Dinman et al, 1991;Tzeng et al, 1992); and in plant luteoviruses and enamoviruses, including beet western yellows virus (Garcia et al, 1993;Miller et al, 1996;Kim et al, 1999). A prominent exception to this general rule is in HIV-1, where a simple 3 H hairpin appears to stimulate -1 frameshifting at the gag-pol junction , but only 3 to 5-fold above that obtained for this rather ef®cient slippery sequence (U UUU UUA) alone (Jacks et al, 1988b;Bidou et al, 1997).…”

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“…The triple-base contacts in the structure show a preference for adenosine residues in loop 2, and appear to be sequence specific. Mutations in other pseudoknots that invert base pairs in stem 1 4,5,7 or eliminate the high adenosine content in loop 2 (ref. 12) are reported to have adverse effects on frameshifting.…”

“…The mature products are later obtained by processing the poly-protein precursor 1 . The -1 frameshifting is found not only in retroviruses [3][4][5] but also in coronaviruses 6 , in yeast 7 and in plant viruses 8 , as well as in bacterial systems 1 . Frameshifting levels can range from 1% to over 30% in different systems to produce gene products in a functionally appropriate ratio.…”

“…Loop 1 and loop 2 are unpaired regions that cross the major groove of stem 2 and the minor groove of stem 1, respectively 10 . Many mutational studies have been performed on frameshifting pseudoknots to correlate structure and function [4][5][6][7] , and certain mutations at the junction of the two stems or loop regions can have deleterious effects on frameshifting efficiencies 11,12 . These findings suggest that there are special structural features involving tertiary interactions in a frameshifting pseudoknot that are important for function.…”

Viruses of Fungi and Protozoans: Is Everyone Sick?

“…The Pol protein is translated as a Cap-Pol fusion product by a frameshift event Dinman et al, 1991). The L 1 Cap-Pol frameshift is similar to frameshifting in the retroviruses, both in site specificity and in RNA secondary and tertiary structure requirements (Dinman et al, 1991;Tzeng et al, 1992).…”