Histidine-tagged RNA polymerase: dissection of the transcription cycle using immobilized enzyme.
A stretch of six histidine residues (His6) has been genetically fused to the C terminus of the beta' polypeptide of Escherichia coli RNA polymerase. The His6-tagged beta' subunit assembles into RNA polymerase molecules which perform all vital in vivo functions and behave qualitatively normally in vitro. The His6 tag permits rapid purification of the enzyme directly from crude cell extracts or from an in vitro reconstitution reaction by adsorption to Ni(2+)-chelating agarose resin, followed by elution with imidazole. The enzyme bound to the matrix remains transcriptionally active. The immobilized enzyme can withstand repeated buffer changes without substantial activity loss and permits controlled stepwise 'walking' of the transcriptional complex along the DNA template, and isolation of defined intermediates in the transcription cycle. The immobilized RNA polymerase provides a powerful experimental system for structural and functional analysis of RNA polymerase and its interaction with regulatory factors.
Kashlev
M
M
Public Health Research Institute, New York, NY 10016.
Martin
E
E
Polyakov
A
A
Severinov
K
K
Nikiforov
V
V
Goldfarb
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004790:Enzyme Induction; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006639:Histidine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009532:Nickel / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016324:Sequence Tagged Sites; D013698:Templates, Genetic; D015533:Transcriptional Activation; D014544:Uridine Triphosphate / Q000378:metabolism
D004269:DNA, Bacterial; D015226:Dinucleoside Phosphates; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; C013907:cytidylyl adenosine; D006639:Histidine; D009532:Nickel; D012321:DNA-Directed RNA Polymerases; D014544:Uridine Triphosphate