Zinc ion effects on individual Ssp DnaE intein splicing steps: regulating pathway progression.
Use of the naturally split, self-splicing Synechocystis sp. PCC6803 DnaE intein permits separate purification of the N- and C-terminal intein domains. Otherwise spontaneous intein-mediated reactions can therefore be controlled in vitro, allowing detailed study of intein kinetics. Incubation of the Ssp DnaE intein with ZnCl(2) inhibited trans splicing, hydrolysis-mediated N-terminal trans cleavage, and C-terminal trans cleavage reactions. Maximum inhibition of the splicing reaction was achieved at equal molar concentrations of ZnCl(2) and intein domains, suggesting a 1:1 metal ion:intein binding stoichiometry. Mutation of the (+)1 cysteine residue to valine (C(+)1V) alleviated the inhibitory effects of ZnCl(2). Valine substitution in the absence of ZnCl(2) blocked trans splicing and decreased C-terminal cleavage kinetics in a manner similar to that of the native (+)1 cysteine in the presence of ZnCl(2). These data are consistent with Zn(2+)-mediated inhibition of the Ssp DnaE intein via chelation of the (+)1 cysteine residue. N-Terminal trans cleavage can occur via both spontaneous hydrolysis and nucleophilic (e.g., DTT) attack. Comparative examination of N-terminal cleavage rates using amino acid substitution (C(+)1V) and Zn(2+)-mediated inhibition permitted the maximum contribution of hydrolysis to overall N-terminal cleavage kinetics to be determined. Stable intermediates consisting of the associated intein domains were detected by PAGE and provided evidence of a rapid C-terminal cleavage step. Acute control of the C-terminal reaction was achieved by the rapid reversal of Zn(2+)-mediated inhibition by EDTA. By inhibiting both the splicing pathway and spontaneous hydrolysis with Zn(2+), reactants can be diverted from the trans splicing to the trans cleavage pathway where DTT and EDTA can regulate N- and C-terminal cleavage, respectively.
Nichols
Nicole Magnasco
NM
New England Biolabs, Inc., Beverly, Massachusetts 01915-5599, USA.
Benner
Jack S
JS
Martin
Deana D
DD
Evans
Thomas C
TC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002614:Chelating Agents / Q000494:pharmacology; D002686:Chitin / Q000235:genetics / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000235:genetics; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D019154:Protein Splicing / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism; D020040:Trans-Splicing / Q000187:drug effects*; D015032:Zinc / Q000494:pharmacology*
D002614:Chelating Agents; D017931:DNA Primers; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D002686:Chitin; D013879:Thioredoxins; D004492:Edetic Acid; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D015032:Zinc; D004229:Dithiothreitol