Zinc inhibition of protein trans-splicing and identification of regions essential for splicing and association of a split intein

Two important aspects of protein splicing were investigated by employing th e trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803. Fi rst, we demonstrated that both protein splicing and cleavage at the N-termi nal splice junction were inhibited in the; presence of zinc ion. The trans- splicing reaction was partially blocked at a concentration of 1-10 muM Zn2 and completely inhibited at 100 muM Zn2+; the inhibition by zinc was rever sed in the presence of ethylenediaminetetraacetic acid. We propose that ina ctivation of Cys(160) at the C-terminal splice junction by the chelation of zinc affects both the N-S acyl rearrangement and the transesterification s teps in the splicing pathway. Furthermore, in vivo and in vitro assays were established for the determination of intein residues and regions required for splicing or association between the N- and C-terminal intein halves. N- terminal truncation of the intein C-terminal segment inhibited both splicin g and association activities, suggesting this region is crucial for the for mation of an interface between the two intein halves. The replacement of co nserved residues in blocks B and F with alanine abolished splicing but allo wed for association. This is the first evidence showing that the conserved residues in block F are required for protein splicing.