Studies of the binding of Escherichia coli RNA polymerase to DNA. I. The role of sigma subunit in site selection.

Abstract A membrane filter assay has been devised to study the binding of Escherichia coli RNA polymerase to DNA. RNA polymerase is quantitatively retained on nitrocellulose filters, and DNA bound in complexes between RNA polymerase and labeled T7 DNA is retained with an efficiency of up to 70%. The attachment of a single RNA polymerase molecule to a molecule of T7 DNA is sufficient to cause retention of the DNA molecule on the filter. Using the filter assay, it has been shown that both RNA polymerase holoenzyme and core polymerase bind to T7 DNA. The interaction of RNA polymerase holoenzyme with T7 DNA leads to two classes of complex. Approximately eight polymerase molecules can bind to each T7 DNA molecule to form a highly stable complex (Kassoc. = 1012 to 1014 m−1). In addition, there are many other sites on the DNA molecule at which a weak complex can be formed (Kassoc. = 108 to 108 m−1). The majority of holoenzyme molecules bound in the highly stable complex are thought to be bound at or near the T7 early promoter region, while the weak binding sites are thought to reflect a low, general affinity of holoenzyme for other regions of the DNA helix. In contrast, only a single major class of binding sites is found on T7 DNA for core polymerase (Kassoc. = 2 × 1011 m−1) and there are many such sites on the T7 genome. These sites are interpreted as reflecting a high general affinity of core polymerase for all regions of the DNA helix. It is concluded that RNA polymerase can exist in two conformational states, one programmed for promoter binding and initiation (holoenzyme) and the other designed for RNA chain elongation (core polymerase). Sigma subunit may function in site selection primarily to switch the enzyme reversibly between these two states.