Electron microscope studies of transient complexes formed between Escherichia coli RNA polymerase holoenzyme and T7 DNA.

Abstract

EM was used to study the formation of random complexes between E. coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and a promoterless fragment (Mbo I-C) of bacteriophage T7 DNA, and to determine the location of the polymerase molecules bound at 0.degree. C to the promoter-containing (Hinf)1100 of the same DNA. The value of the Ka of random binding is about 3 .times. 104 M-1 when the enzyme is slowly diluted from its storage condition and is incubated with DNA for up to 2 min at 37.degree. C. If dilution is rapid and occurs in a single step, or if incubation extends beyonds 5 min, a substantial portion of RNA polymerase is converted to a form that binds randomly with a much greater affinity (about 108 M-1). Hence true random binding by RNA polymerase holoenzyme is much weaker than previously thought. Great caution is required in assessing the extent of random binding where damage to the enzyme may occur. When RNA polymerase holoenzyme is incubated at 0.degree. C with promoter-containing fragment (Hinf)1100, complexes form at the same promoter sites utilized at 37.degree. C, although the highly stable open promoter complex is not formed under these conditions. The extent of binding is reduced as compared to promoter complexes formed at 37.degree. C. This gives direct evidence for formation of complexes with promoter sites that have properties of the hypothetical closed complexes formed between RNA polymerase and duplex DNA.