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

e slippery sequence and stimulatory element. For the IBV frameshift signal, this is known to be the case; altering the natural spacing distance (6 nt) between pseudoknot and slippery sequence (UUUA AAC) by a single nucleotide either way has a 2-fold inhibitory effect on frameshifting, and a 5-fold reduction is seen when 2 nt are added to or removed from the spacer (12, 37) . Regarding AON-stimulated frameshifting, current evidence also supports a requirement for appropriate spacing. In the studies of Howard and colleagues (30), efficient frameshifting was seen with spacer lengths from À1 to 5 nt, with an optimum at 3 nt (as used in this study). Given the observation in the present work of AON-stimulated À1 and À2 FS, it was important to ascertain the optimal spacing distance for the two events. To do this, we modified pFSHIV-AON stopAll to generate plasmids (pFSHIV-AON spacer series) with spacers varying in length from 0 to 9 nt (Figure 6 panel A). In addition, for ease of comparison, a single base was added or removed from the mRNA downstream of the AON-binding region such that the size of the various translation products (stop, À1 FS, À2 FS) was maintained between constructs ( Figure 6 panel B) . In translations of the pFSHIV-AON spacer series, with added 25MO or 25OMe (at 1 mM in this experiment), we observed discrete peaks of frameshifting, with the overall pattern essentially the same, except that frameshifting was more efficient with 25OMe. The À1 FS product was evident across a broad range, spanning spacer lengths 0-7 nt, but with two optima, at 0 nt and at 3 nt, (particular noticeable in the 25MO titration). The À2 FS product had an optimal spacing of 2 nt and was evident over the range 1-5 nt with 25OMe, but more discrete with 25MO (2-3 nt). In the absence of added AON, both frameshift products were detectable although at low levels, and unsurprisingly, there were no spacer effects.