Intermediates in transcription initiation from the E. coli lac UV5 promoter

Abstract

We have used nondenaturing polyacrylamide gel electrophoresis to separate intermediates in transcription initiation that result from action of E. coli RNA polymerase on the lac UV5 promoter. The resolved gel complexes are characterized by DNAase I footprinting, protein subunit content, RNA content, and transcription ability. There are two “open” complexes, whose equilibrium ratio is a function of temperature; they differ in their ability to escape abortive cycling, but not in their DNAase I footprints. We find three “initiated” complexes, containing RNA chains at least 11 nucleotides long, and lacking the o subunit of RNA polymerase. These experiments provide a detailed view of the early initiation steps and their thermal regulation at the E. coli lac promoter.