Retention of transcription initiation factor σ 70 in transcription elongation: single-molecule analysis

We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and σ70 contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, ∼70%–90%, of early elongation complexes retain σ70 and that a determinant for σ70 recognition in the initial transcribed region increases σ70 retention in early elongation complexes. The results establish that a significant fraction, ∼50%–60%, of mature elongation complexes retain σ70 and that a determinant for σ70 recognition in the initial transcribed region does not appreciably affect σ70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain σ70, the half-life of σ70 retention is long relative to the timescale of elongation, suggesting that some complexes may retain σ70 throughout elongation.