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

We showed that the a-L-fucosidase gene from the crenarchaeon Sulfolobus solfataricus is putatively expressed by programmed À1 frameshifting (24) . This gene, named fucA1, is organized in the open reading frames (ORFs) SSO11867 and SSO3060 of 81 and 426 amino acids, respectively, which are separated by a À1 frameshifting in a 40 base overlap ( Figure 1A ). We have reported previously that the region of overlap between the two ORFs had the characteristic features of the genes expressed by programmed À1 frameshifting including a slippery heptanucleotide A-AAA-AAT (codons are shown in the zero frame) flanked by a putative stem-loop and the rare codons CAC ( Figure 1A ) resembling the prokaryotic stem-loops/hairpins and the Shine-Dalgarnolike sites (24) . We showed that the frameshifting, obtained by mutating by site-directed mutagenesis the fucA1 gene exactly in the position predicted from the slippery site, produced a full-length gene, named fucA1 A , encoding for a polypeptide of 495 amino acids ( Figure 1B ). This mutant gene expressed in Escherichia coli a fully functional a-L-fucosidase, named Ssa-fuc, which was thermophilic, thermostable and had an unusual nonameric structure (24, 25) . More recently, we determined the reaction mechanism and the function of the residues of the active site of the mutant enzyme (26, 27) .