Transcription by T7 RNA polymerase is not zinc-dependent and is abolished on amidomethylation of cysteine-347.
T7 RNA polymerase has been purified to homogeneity from an overproducing clone of Escherichia coli containing pAR1219. Preparations have a zinc content as low as 0.01 mol/mol of enzyme and a high specific activity, 300 000-500 000 units/mg. There are no intrinsic zinc sites. Furthermore, extrinsic Zn2+ does not function as an activator. Supplementation of the assay mix with up to 5 mM ethylenediaminetetraacetic acid has little effect on activity while added Zn2+ is strongly inhibitory at concentrations above 10 microM. This monomeric RNA polymerase is not a zinc metalloenzyme, unlike its multimeric bacterial counterparts. Titration of the urea-denatured protein with 5,5'-dithiobis(2-nitrobenzoic acid) reveals that all 12 Cys residues are present in the free sulfhydryl form, 5 of which are readily accessible to reagent in the native enzyme. More preferential labeling of the sulfhydryls can be achieved with low concentrations of [14C]iodoacetamide, where inactivation of the enzyme proceeds with incorporation of approximately 1.2 mol of [14C]iodoacetamide/mol of polymerase. Amidomethylation primarily occurs at Cys-347, with lesser reaction at Cys-723 and Cys-839. Cys-347 and Cys-723 are in segments of the primary sequence containing numerous basic residues. These same segments have previously been implicated in promoter binding, suggesting that both residues are located within or near the active site region.
King
G C
GC
Martin
C T
CT
Pham
T T
TT
Coleman
J E
JE
D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007460:Iodoacetamide / Q000494:pharmacology*; D007461:Iodoacetates / Q000494:pharmacology*; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects*; D014357:Trypsin; D015032:Zinc / Q000494:pharmacology*
D000596:Amino Acids; D007461:Iodoacetates; D010446:Peptide Fragments; D004492:Edetic Acid; D014357:Trypsin; D015032:Zinc; D003545:Cysteine; D007460:Iodoacetamide