Inactivation and destruction by KMnO4 of Escherichia coli RNA polymerase open transcription complex: recommendations for footprinting experiments.
Potassium permanganate oxidation of pyrimidine residues in single-stranded DNA is commonly used in footprinting studies on formation of open transcription complex (RPo) by RNA polymerases (RNAP) at cognate promoters. Our own experience and literature search led us to conclude that KMnO4 doses often used in such studies might cause multiple-hit oxidation of promoter DNA and oxidative damage to RNAP in RPo and lead to false interpretation of footprints. We have therefore studied as a function of KMnO4 dose (i) transcription activity of RPo formed by Escherichia coli RNAP at a model cognate promoter Pa and (ii) RPo's structural integrity, by gel electrophoresis and footprinting assays. Kinetics of formation of this complex and melting of DNA in the transcription bubble region were thoroughly characterized by us previously. Here we show that (i) RPo becomes completely inactivated at oxidant doses much lower than those needed to cause a detectable footprint of the melted DNA region, (ii) footprinting patterns of the melted promoter region remain practically unaffected by RNAP oxidation within a range of low oxidant doses causing single-hit oxidation of DNA, and (iii) at higher oxidant doses, corresponding to multiple-hit DNA oxidation, the gross structure of RPo changes progressively until its complete collapse and dissociation into constituent components, so that only approximate interpretation of the footprinting data for the melted DNA region is possible. A protocol for accurate RPo footprinting with low single-hit KMnO4 doses and interpretation of the footprinting data in terms of kinetics of oxidation of pyrimidine residues in promoter DNA is recommended.
Loziński
Tomasz
T
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warszawa, Poland.
Wierzchowski
Kazimierz L
KL
D001483:Base Sequence; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000187:drug effects*; D003627:Data Interpretation, Statistical; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016877:Oxidants / Q000494:pharmacology*; D011196:Potassium Permanganate / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects*
D016877:Oxidants; D011196:Potassium Permanganate; D012321:DNA-Directed RNA Polymerases