Abstract
Production of a messenger RNA proceeds through sequential stages of transcription initiation, transcript elongation and termination; all three stages are regulated to control gene expression. As RNA polymerases (RNAPs) transition to elongation, they lose association with one or more initiation protein factors required to recognize transcription start sites, and they acquire proteins specifically involved in regulating transcript elongation and termination. Escherichia coli σ70 is an initiation factor. Previous studies reached conflicting conclusions about the extent to which σ70 is retained after elongation begins, and the rate of its release from RNAP actively undergoing transcript elongation has not been directly measured. We used multiwavelength single-molecule fluorescence co-localization microscopy to observe the association of σ70 with RNAP during initiation and throughout the elongation of a 2,400 nucleotide transcript. We observe that a majority of RNAP molecules lose σ70 concommitant with promoter escape, while a subpopulation of elongation complexes retain σ70 throughout elongation of the full transcript. The fraction of σ70 retained on elongation complexes is influenced by the sequence of the initial transcribed region. The presence of σ70 on elongation complexes far downstream of the promoter is expected to substantially alter their regulation by affecting transcription pausing, termination, and possibly re-initiation.