Excision de la region du DNA liee a la RNA polymerase in vitro

Abstract

After synthesis of short, nascent oligonucleotide in the presence of (32P)DNA, GTP, CTP, UTP and 3'dATP, one can excise with deoxyribonuclease a ternary complex of RNA polymerase, protected DNA and oligonucleotide, while the enzyme simply bound to the template is removed by increasing the ionic strength. This ternary complex is retained on nitrocellulose membranes. On polyacrylamide gel electrophoresis it migrates faster than RNA polymerase alone. The protected portion of the DNA is constituted of about 75 nucleotides. It might represent the sites for RNA initiation.