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 the trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803. First, we demonstrated that both protein splicing and cleavage at the N-terminal splice junction were inhibited in the presence of zinc ion. The trans-splicing reaction was partially blocked at a concentration of 1-10 microm Zn(2+) and completely inhibited at 100 microm Zn(2+); the inhibition by zinc was reversed in the presence of ethylenediaminetetraacetic acid. We propose that inactivation of Cys(160) at the C-terminal splice junction by the chelation of zinc affects both the N-S acyl rearrangement and the transesterification steps 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 splicing and association activities, suggesting this region is crucial for the formation of an interface between the two intein halves. The replacement of conserved residues in blocks B and F with alanine abolished splicing but allowed for association. This is the first evidence showing that the conserved residues in block F are required for protein splicing.
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New England Biolabs, Inc., Beverly, Massachusetts 01915, USA.
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D000095:Acetolactate Synthase / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D017124:Conserved Sequence; D000458:Cyanobacteria / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015151:Immunoblotting; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D019154:Protein Splicing / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000494:pharmacology*
D001426:Bacterial Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000095:Acetolactate Synthase; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D015032:Zinc