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

In an attempt to determine the amino-acid sequence of the presumed À2 FS product, we carried out a large-scale in vitro translation of a tagged version of pFSHIV-AON with optimal (2 nt) spacer, but probably due to the low productivity of RRL, it proved impossible to isolate material of sufficient purity and yield for unambiguous N-terminal sequence determination by Edman degredation. A similar problem was encountered with a tissue culture expression system in which frameshifting was dependent upon co-transfection of 25OMe. However, it proved possible to purify sufficient +1/À2 product for MS analysis when a frameshift cassette of U 6 A, 7 nt spacer and stem-loop stimulator was expressed as an N-terminal fusion with eukaryotic green fluorescent protein (eGFP). This plasmid (pFSeGFP-N2, see section 'Materials and Methods') was transfected into 293 T cells and the À2 FS product purified by affinity chromatography (utilizing a GFP binding matrix), gel electrophoresis and band excision. Following digestion with trypsin, resultant peptides were analysed by MALDI mass fingerprinting and subsequent tandem mass spectrometry (ESIMS-MS). Peptides corresponding to 77% of the predicted fusion protein were identified and the sequence spanning the frameshift region was determined as LNFLYE, indicating À2 FS (Figure 8 ; raw data in Supplementary Figure S3 ). The peptide fingerprint data were scanned for other possible events, including the aforementioned P-site+1 FS (generating LNFYE), or sequential À1 ribosomal frameshifts in consecutive elongation cycles (with the first on G AAU UUU and the second on U UUU UUA; generating LNFFYE) but no matches were present.