A Study of Unwinding of DNA and Shielding of the DNA Grooves by RNA Polymerase by Using Methylation with Dimethylsulphate

Abstract

The dimethylsulphate method has been used to study the complexes of RNA polymerase (Escherichia coli) with DNA of T7 phage, poly[d(A--T)] and fragments of calf thymus DNA protected against DNase digestion by RNA polymerase. The binding of RNA polymerase to DNA significantly increases the formation of 1-methyl-adenine produced by methylation of the single-stranded DNA region, diminishes by about 10% the formation of 3-methyl-adenine by methylation within the minor groove and does not affect the formation of 7-methyl-guanine by methylation within the major DNA groove. The presence of nascent RNA decreases the formation of 1-methyl-adenine in DNA of the complex by about 30%. The initiation of RNA synthesis or RNA synthesis itself does not influence the methylation of the major groove but shielding of the minor groove increases by about twice as much. These results suggest that RNA polymerase, upon binding, breaks Watson-Crick base-pairing in a DNA region of about 15-base-pairs long, that nascent RNA forms a duplex with DNA of about 10-base-pairs long; and that the enzyme weakly interacts with DNA along its grooves and preferentially makes contacts with the minor groove.