Initiation of transcription by Escherichia coli RNA polymerase from supercoiled and non-supercoiled bacteriophage PM2 DNA

Abstract

Native bacteriophage PM2 DNA, a circular double-helical DNA with —50 superhelical twists, and derivatives of PM2 DNA containing no superhelical twists are compared for their ability to form stable binary complexes with Escherichia coli RNA polymerase. The number of enzyme molecules that can form such complexes, as measured by the number of chains initiated with ATP and GTP in the presence of saturating amounts of enzyme and using heparin to inactivate the unbound enzyme, is 16 per molecule of native PM2 DNA but only about two per molecule of closed non-supercoiled PM2 DNA. In each case equal numbers of chains are initiated with GTP and ATP. The reactivity of the sites on the two forms are also different; the rate of formation and the stability of the complexes are both lower on the non-supercoiled derivatives than on native PM2 DNA. The derivatives also contain a more significant proportion of sites that bind RNA polymerase tightly in an inactive state than native PM2 DNA. Thus, even with excess DNA (so that the number of sites is not limiting) and with sufficient preincubation to form the complexes, the non-supercoiled forms of DNA are still less active templates than native PM2 DNA. On the other hand, the active sites on the two DNAs are similar in one respect; the rates of RNA chain initiation by RNA polymerase bound to these sites are the same.