The gene of an archaeal a-l-fucosidase is expressed by translational frameshifting

Cobucci-Ponzano, Beatrice; Conte, Fiorella; Benelli, Dario; Londei, Paola; Flagiello, Angela; Monti, Maria; Pucci, Piero; Rossi, Mosè; Moracci, Marco

The wild-type fucA1 gene, expressed in E.coli as a GST-fused protein, produced trace amounts of a-fucosidase activity (2.3 · 10 À2 units mg À1 after removal of GST), suggesting that a programmed À1 frameshifting may occur in E.coli (24) . The enzyme was then purified by using the GST purification system and analysed by SDS-PAGE revealing a major protein band ( Figure 2A ). The sample and control bands were excised from the gel, digested in situ with trypsin and directly analysed by matrix-assisted laser desorption/ ionization mass spectrometry (MALDIMS). As shown in Figure 2B and C, both spectra revealed the occurrence of an identical mass signal at m/z 1244.6 corresponding to a peptide (Peptide A) encompassing the overlapping region of the two ORFs. This result was confirmed by liquid chromatography online tandem mass spectrometry (LCMSMS) analysis of the peptide mixtures. The fragmentation spectra of the two signals showed the common sequence Asn-Phe-Gly-Pro-Val-Thr-Asp-Phe-Gly-Tyr-Lys in which the amino acid from the ORF SSO11867 is underlined. These results unequivocally demonstrate that the protein containing the Peptide A is produced in E.coli by a frameshifting event that occurred exactly within the slippery heptamer predicted from the analysis of the DNA sequence in the region of overlap between the ORFs SSO11867 and SSO3060 ( Figure 1A) .