MUTATIONAL ANALYSIS OF THE RNA PSEUDOKNOT INVOLVED IN EFFICIENT RIBOSOMAL FRAMESHIFTING IN SIMIAN RETROVIRUS-1

Mutational effects on frameshifting efficiency of the RNA pseudoknot i nvolved in ribosomal frameshifting in simian retrovirus-1 (SRV-1) have been investigated, The primary sequence and the proposed secondary st ructure of the SRV-1 pseudoknot are similar to those of other efficien t frameshifting pseudoknots in mouse mammary tumor virus (MMTV) and fe line immunodeficiency virus (FIV), where an unpaired adenine nucleotid e intercalates between stem 1 and stem 2. In SRV-1 pseudoknot, the ade nine nucleotide in between stem 1 and stem 2 has a potential to form a n A.U base pair with the last uridine nucleotide in the loop 2, result ing in a continuous A-form helix with coaxially stacked stem 1 and ste m 2, To test whether this A.U base pairing and coaxial stacking of ste m 1 and stem 2 is absolutely required for efficient frameshifting in S RV-1, a series of mutants changing this potential A.U base pair to eit her G.C base pair or A.A, A.G, A.C, G.A, G.G mismatch is generated, an d their frameshifting efficiencies are investigated in vitro using rab bit reticulocyte lysate translation assay, The frameshifting abilities of these mutant-pseudoknots are similar to that of the wild-type pseu doknot, suggesting that the A.U base pair in between stem 1 and stem 2 is not necessary to promote efficient frameshifting in SRV-1, These r esults reveal that coaxial stacking of stem 1 and stem 2 with a Watson -Crick A.U base pair in between two stems is not a required structural feature of the pseudoknot for promoting efficient frameshifting in SR V-1, Our mutational data suggest that SRV-1 pseudoknot adopts similar structural features common to other efficient frameshifting pseudoknot s as observed in MMTV and FIV.