Spacer-length dependence of programmed -1 or -2 ribosomal frameshifting on a U(6)A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting

Lin, Zhaoru; Gilbert, Robert J. C.; Brierley, Ian

Studies of programmed À1 ribosomal frameshifting have focused predominantly on stem-loop and pseudoknotdependent signals (reviewed in 3, 4) . From these investigations, a plausible model of frameshifting has emerged which posits a critical contribution of the stimulatory RNA in compromising the activity of the proposed intrinsic helicase activity of the 80S ribosome, located at the mRNA entry channel (15, 28) . A failure to unwind the stimulatory RNA appropriately during the elongation cycle would potentially compromise frame maintenance through the generation of tension in the mRNA, effectively pulling the mRNA in a 3 0 -direction while promoting breakage of the tRNA anticodon:codon interaction and realignment of the tRNA in the 5 0 -(À1) direction. In support of this model, ribosomes are known to pause at frameshift-stimulatory Figure 7 . Continued variants of the two plasmids in which the first position of the slippery sequence was changed to G, A or C. (E) Messenger RNAs derived from Bam HI-cut pFSHIV-PK spacer variants were translated in RRL and products analysed and quantified as in panel C. The numbers above each gel represent the spacer length. The frameshifting efficiency measured for each signal (to the nearest integer) is indicated below the relevant lanes (À1% FS; À2% FS) and takes into account the number of methionines present in each product (nFS, 10; À1 FS, 11; À2 FS, 35) . The asterisk marks the position of the À1 FS product of pFS cass 5 control mRNA (IBV PK) and also an additional product seen in the translation of the pFSHIV-PK 9 nt spacer mRNA. The size of the latter product is consistent with one that would be synthesised following readthrough of the UGA codon the terminates the non-frameshifted product (present in the spacer; see text). stem-loops and pseudoknots, indicating that such structures can act as barriers to elongation (20, 21, 37, (39) (40) (41) (42) (43) . In addition, cryo-EM reconstructions of 80S rabbit ribosomes stalled at the IBV frameshift-promoting pseudoknot have revealed a distorted tRNA in a hybrid A/P-like state, with the anticodon arm bent markedly towards the A-site of the ribosome (22, 44, 45) . In these reconstructions, density that likely corresponds to the pseudoknot is observed at the mRNA entry channel close to the putative 80S helicase. These features are consistent with the model, with the pseudoknot resisting unwinding during eEF2-mediated translocation such that tension builds up in the mRNA, subsequently placing strain on the tRNA and resulting in the adoption of a bent conformation (22) . In mechanical unwinding studies, a functional IBV-based pseudoknot has been shown to be a 'brittle' structure, with a shallow dependence of the unfolding rate on applied force and a slower unfolding rate than component hairpin structures (Green et al., 2008) . This greater mechanical stability and kinetic insensitivity to force is consistent with a role in resistance to unwinding (23) (24) (25) 29) . Indeed, a number of pseudoknot features have been identified that could act in such resistance, such as the unusual topology of stems and loops, the geometry of the junction of the two stems and in some pseudoknots, base triplexes between loop 3 and stem 1 (reviewed in 3, 4) . While some or all of these features are readily identifiable in pseudoknot-stimulatory RNAs, the situation is not so clear-cut for stem-loop stimulatory RNAs. Nuclear magnetic resonance (NMR) analysis of the HIV-1 (46-48) and