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

MALDIMS and LCMSMS analyses of the products in E.coli of the wild-type split gene fucA1 demonstrated that two independent frameshifting events occurred in vivo in the proposed slippery site. In particular, the sequences obtained by LCMSMS demonstrate that peptide A results from a simultaneous backward slippage of both the P-and the A-site tRNAs ( Figure 8A) . Instead, the sequence of peptide B is the result of the re-positioning on the À1 frame of only the P-site tRNA; in fact, the next incorporated amino acid is specified by the codon in the new frame ( Figure 8B) . Therefore, the expression by À1 frameshifting of the wild-type gene fucA1 in E.coli follows the models proposed for ribosomal frameshifting (34) . We confirmed the significance of the slippery heptanucleotide in promoting the programmed frameshifting in vivo by mutating the putative regulatory sequence. The triple mutant fucA1 tm gave no full-length products; presumably, the mutations in both the P-and in the A-site of the slippery sequence dramatically reduced the efficiency of the À1 frameshifting as observed previously in metazoans (35) . This result confirms that the intact slippery sequence in the wild-type gene fucA1 is absolutely necessary for its expression in E.coli. In contrast, surprisingly, the single mutant fucA1 sm showed an even increased frequency of frameshifting (10%) if compared to the wild-type and produced only one polypeptide by shifting specifically in site B. We explained this result observing that the mutation in the P-site of the slippery sequence A-AAA-AAT!A-AAG-AAT created a novel slippery sequence A-AAG identical to that controlling the expression by programmed À1 frameshifting of a transposase gene in E.coli (36) . Therefore, apparently, the single mutation inactivated the simultaneous P-and A-site tRNA re-positioning and, in the same time, fostered the shifting efficiency of the tRNA in the P-site. It is worth noting that, instead, in S.solfataricus, only the simultaneous slippage is effective ( Figure 8B ) and even the single mutation in the slippery sequence of fucA1 sm completely annulled the expression of the gene. This indicates that this sequence is essential in the archaeon and that programmed frameshifting in S.solfataricus and E.coli exploits different mechanisms. Furthermore, since the only difference between the enzymes produced by the frameshifting sites A and B, Ssa-fuc and Ssa-fuc B , respectively, is the stability at 80 C, which is the S.solfataricus physiological temperature, the functionality of Ssa-fuc B in the archaeon appears questionable.