Functional analysis of the SRV-1 RNA frameshifting pseudoknot

Abstract

Simian retrovirus type-1 uses programmed ribosomal frameshifting to control expression of the Gag-Pol polyprotein from overlapping gag and pol open-reading frames. The frameshifting signal consists of a heptanucleotide slippery sequence and a downstream-located 12-base pair pseudoknot. The solution structure of this pseudoknot, previously solved by NMR [Michiels,P.J., Versleijen,A.A., Verlaan,P.W., Pleij,C.W., Hilbers,C.W. and Heus,H.A. (2001) Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting. J. Mol. Biol., 310, 1109–1123] has a classical H-type fold and forms an extended triple helix by interactions between loop 2 and the minor groove of stem 1 involving base–base and base–sugar contacts. A mutational analysis was performed to test the functional importance of the triple helix for −1 frameshifting in vitro. Changing bases in L2 or base pairs in S1 involved in a base triple resulted in a 2- to 5-fold decrease in frameshifting efficiency. Alterations in the length of L2 had adverse effects on frameshifting. The in vitro effects were well reproduced in vivo, although the effect of enlarging L2 was more dramatic in vivo. The putative role of refolding kinetics of frameshifter pseudoknots is discussed. Overall, the data emphasize the role of the triple helix in −1 frameshifting.

Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for −1 ribosomal frameshifting stimulation

“…Previously, an in vitro frameshifting assay was performed with all constructs containing the same slippery sequence (G GGA AAC ) and a 7-nt single-stranded spacer19. A 7-nt spacer (UGAUCGA) is present in SF206, SF209 and SF211, and a slightly different spacer (UAGUUGA) is present in the four remaining constructs.…”

“…1a and Supplementary Fig. S1) whose design was based on the NMR structure19. The structure of SF206 reveals consecutive minor-groove base triples formed between stem 1 and loop 2 (Fig.…”

“…1c). Mutations were designed to disrupt the base triples and the base stacking interactions expected to affect frameshifting efficiencies19. The previous mutational studies19 revealed that SF206 and the six mutants display in vitro frameshifting efficiencies in the range between 21% and 1%, which allows us to investigate the possible coupling effect between stem 1 and loop 2 on thermal and mechanical stabilities of the frameshifting pseudoknots, and the possible correlation with the frameshifting efficiency.…”

“…An mRNA structure located downstream of the slippery sequence separated by a single-stranded spacer (2 to 8 nucleotides (nt) in length) stimulates −1 frameshifting from 0 frame X XXY YYZ to −1 frame XXX YYY Z. The downstream frameshifting stimulatory structure can be a hairpin (stem-loop)61617, a pseudoknot5891012151819, a duplex formed between the mRNA and an antisense oligonucleotide20212223, or a G-quadruplex2425.…”

“…An NMR study revealed that minor-groove base triples form between stem 1 and loop 2 of the pseudoknot18. Extensive mutagenesis assays have revealed that the frameshifting efficiencies are highly dependent on the loop residues in the pseudoknot1819. A coarse-grained simulation suggests a cooperative thermal unfolding of the two stems of the SRV-1 pseudoknot41.…”