The study of DNA-RNA-polymerase complexes by kinetic formaldehyde method.
A modification of the kinetic formaldehyde method has been proposed providing a possibility for locally denatured regions (defects) formed in DNA preincubated with RNA polymerase (in the absence of nucleoside triphosphates) to be detected. This modification consists in a previous fixation of DNA-enzyme complex with small concentrations of formaldehyde, which do not induce formation of defects in DNA alone. The method has been calibrated under the conditions favourable to RNA synthesis. Studies of the effect of the fixation conditions on the number of defects in DNA interacting with RNA polymerase have shown that the number of defects is constant with formaldehyde fixation concentration between 0.05% and 0.3-0.5% and with fixation time between 2 min and 100 min. The dependence of the number of defects in DNA on RNA polymerase concentration at low ionic strength (0.05 M KCl) is presented by a curve with a plateau. From the initial linear part of the curve it has been found that the enzyme bound to DNA as a monomer. At the excess of the enzyme the mean number of nucleotide pairs between defects is 400-500. Increase of ionic strength results in decrease of the number of defects in DNA. The number of defects depends on temperature of preincubation of the complex. There were no defects in DNA at temperatures below 20 degrees C. At temperatures above 30 degrees C the number of defects reaches saturation. A sharp transition occurs in the range of temperatures between 20 degrees C and 30 degrees C. Analysis of the experimental and literature data, concerning the interaction of formaldehyde and amino acid methylol derivatives with DNA bases, leads to the conclusion that the mechanism of the formation of defects in helical DNA most likely consists in its unwinding or sharp weakening upon binding of RNA polymerase, prior to addition of formaldehyde.
D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000201:enzymology; D005557:Formaldehyde / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D011485:Protein Binding
D046911:Macromolecular Substances; D005557:Formaldehyde; D004247:DNA; D012321:DNA-Directed RNA Polymerases