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 full-length a-fucosidase produced by the À1 frameshifting event in site B (Ssa-fuc B ), resulting from the single P-site slippage has different properties from Ssa-fuc, whose sequence arises from the simultaneous P-and A-site slippage, we prepared the enzyme by site-directed mutagenesis. The slippery sequence in fucA1 A-AAA-AAT was mutated in A-AAG-AAG-T where mutations are underlined. The new mutant gene was named fucA1 B . The first G, producing the conservative mutation AAA!AAG, was made to disrupt the slippery sequence and hence reducing the shifting efficiency. The second G was inserted to produce the frameshifting that results in the amino acid sequence of Peptide B. Therefore, the sequence of the two full-length mutant genes fucA1 A and fucA1 B differs only in the region of the slippery sequence: A-AAG-AAT-TTC-GGC and A-AAG-AAG-TTC-GGC, respectively (the mutations are underlined, the nucleotides in boldface were originally in the À1 frame) ( Table 1) .