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

An interesting aspect of U 6 A-mediated frameshifting was the occurrence of À1 FS at shorter spacing distances (e.g. 3-5 nt in pFSHIV-SL), albeit at a lower efficiency (e.g. Figure 7 panel C). This could be a result of frameshifting one codon earlier on the mRNA, where the stimulatory RNA effect would be most apparent. At this position in pFSHIV-SL, the sequence G AAU UUU is present, which by chance is compatible (in principle) with tandem À1 FS. However, this sequence is also present in pFSIBV-SL, where À1 FS at spacers of 3-5 nt was less evident (Figure 7 panel C) . An alternative explanation is that a single P-site tRNA slip is occuring on U 6 A (and to a lesser extent on UUUAAAC) at the shorter spacing distances, which is more favoured with P-site tRNA Phe (U UUU UUA) than tRNA Leu (U UUA AAC). An unexpected observation was the appearance of a third 'recoding' product in translations of pFSHIV-PK with a 9 nt spacer. This product (asterisked in Figure 7 panel E) corresponds to readthrough of the UGA codon immediately downstream of the slippery sequence. Thus at an appropriate spacing distance, a frameshift-promoting pseudoknot can induce stop-codon readthrough. That this was observed at a spacing of 9 nt is consistent with the longer spacing requirements generally observed for those naturally occurring readthrough signals that have a stimulatory RNA component (38) . . The effect of slippery sequence-stem-loop (SL) and slippery sequence-pseudoknot (PK) spacing on À1 and À2 FS. (A) The spacer of pFSHIV-SL and pFSIBV-SL was changed from three to nine nucleotides as indicated. In mRNAs derived from these plasmids, frameshifting is stimulated by a SL structure (whose length and base-pair composition is identical to the stacked stems of the minimal IBV PK; see text). Also shown is a diagrammatic representation of potential translation products of pFSHIV-SL mRNAs and predicted molecular masses. Note that zero frame ribosomes terminate at the stop-codon in the spacer in all cases. As previously, the sizes of the encoded frameshift products were normalized by appropriate deletion of bases downstream of the SL. Slippery sequence variants of pFSHIV-SL were also prepared in which the U 6 A sequence was changed to those indicated. (B) The spacer of pFSHIV-PK was changed from three to nine nucleotides as indicated. In these plasmids, frameshifting is stimulated by the minimal IBV PK (12) . As with pFSHIV-SL and derivatives, zero frame ribosomes terminate at the stop-codon in the spacer in all cases, but note that the reading frames of frameshifted ribosomes differ. (C) Messenger RNAs derived from Nco I-cut pFSIBV-SL and pFSHIV-SL spacer variants were translated in RRL and products analysed and quantified as in the legend to Figure 3 . 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, 10; À2 FS, 11). (D) Messenger RNAs derived from Nco I-cut pFSHIV-SL variants with 7 nt or 8 nt spacers were translated in RRL and products analysed and quantified as in panel C. Also tested were