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 determine whether, and with what efficiency, the À1 frameshifting could be performed by S.solfataricus ribosomes, mRNAs obtained by in vitro transcription of the cloned wild-type fucA1 gene and the mutants thereof were used to program an in vitro translation system prepared as described by Condò et al. (28) . To this aim, a promoter of T7 polymerase was inserted ahead of the gene of interest to obtain RNA transcripts endowed with the short 5 0untranslated region of 9 nt observed for the natural fucA1 mRNA (24) . Autoradiography of an SDS-PAGE of the translation products (Figure 7 ) revealed that the wild-type fucA1 transcript produced a tiny but clear band whose molecular weight corresponded to that of the full-length Ssa-fuc obtained by site-directed mutagenesis (24); the latter was translated quite efficiently in the cell-free system in spite of being encoded by a quasi-leaderless mRNA. Judging from the relative intensity of the signals given by the translation products of the wild-type fucA1 and the full-length mutant fucA1 A , the efficiency of the À1 frameshifting in the homologous system was $10%. No signals corresponding to the polypeptides expected from the separated ORFs SSO11867 and SSO3060 (9.6 and 46.5 kDa, respectively) were observed. However, it should be noted that the product of SSO11867, even if synthesized, is too small to be detected in the gel system employed for this experiment. The larger product of ORF SSO3060, on the other hand, is certainly absent. These data unequivocally demonstrate that the ribosomes of S.solfataricus can decode the split fucA1 gene by programmed À1 frameshifting with considerable efficiency producing a full-length polypeptide from the two ORFs SSO11867 and SSO3060.