Effects of λ development on template specificity of Escherichia coli RNA polymerase

Abstract

Treatment of bacterial cell extracts with micrococcal nuclease and subsequent inhibition of nuclease activity by addition of EDTA and 3′, 5′-thymidine diphosphate was used to achieve DNA-dependent RNA synthesis without resorting to enzyme fractionation. Following heat induction of λ CI857 lysogens or infection by λ, a 4–5-fold selective stimulation of transcription of λ DNA by nuclease-treated crude extracts was observed. Extracts isolated from non-lysogenic bacteria, uninduced lysogens, and N − mutants copied λ and Escherichia coli DNA templates almost equally; however, mutation of the Q gene did not affect the λ-dependent transcription activity. The λ-dependent activity is sensitive to inhibition by rifampicin, and copurifies with the E. coli enzyme through (NH 4) 2SO 4 fractionation and low-salt glycerol gradient centrifugation steps. However, the altered activity is highly labile, and is lost following high-salt glycerol gradient centrifugation. Experiments employing various deletion mutants suggest that the genes involved in determining this activity are located between exo and CIII.