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

To test if the scarce amounts of the a-fucosidase in S.solfataricus extracts was the result of reduced expression at transcriptional level, we performed a northern blot analysis of total RNA extracted from cells grown either on YSM or YGM media. We could not observe any signal by using probes matching the 3 0 of the ORF SSO3060 (data not shown). These results suggest that fucA1 produced a rare transcript; therefore, we analysed the level of mRNA by RT-PCR and by real-time PCR. A band corresponding to the region of overlap between the ORFs SSO11867 and SSO3060 was observed in the RNA extracted from cells grown on YSM 2 mg) , the product of the gene fucA1 sm (2 mg), and Ssa-fuc (4 mg). The bands with faster electrophoretic mobility result from the proteolytic cleavage of the full-length protein (25) . (B) Partial MALDIMS spectrum of the tryptic digest from mutant fucA1 sm expressed in E.coli. The mass signal corresponding to peptide C encompassing the overlapping region is indicated. (C) LCMSMS analysis of peptide C. The amino acid sequence inferred from fragmentation spectra is reported. (D) Western blot of E.coli cellular extracts expressing fucA1 A , the wild-type fucA1, fucA1 sm and fucA1 tm genes (Materials and Methods). The blot was probed with anti-GST antibodies. (E) Western blot of partially purified protein samples expressed in E.coli fused to GST from wild-type and mutant fucA1 genes. Cellular extracts were loaded on GST-Sepharose matrix. After washing, equal amounts of slurries (30 ml of 300 ml) were denaturated and loaded on a 8% SDS-PAGE. Extracts of E.coli cells expressing the parental plasmid pGEX-2TK were used as the negative control (pGEX). The blot was probed with anti-Ssa-fuc antibodies. and YGM media, demonstrating that under these conditions the two ORFs were co-transcribed ( Figure 6A) .