An Abortive Isomerization Branch in the Transcription Initiation Pathway At a σ54 Promoter As Revealed By Single Molecule Fluorescence Microscopy

Abstract

Regulated transcription initiation is a complex process that involves multiple protein factors and a series of polymerase-DNA complexes that are intermediates in the reaction. This complexity presents a significant challenge for ensemble experiments that aim to elucidate the reaction pathway. We here report the kinetic mechanism of initiation at the σ54 promoter of the glnALG operon in Salmonella typhimurium. This prototypical activator-dependent promoter is regulated by nitrogen stress. To circumvent the complexity of ensemble analysis, we used multi-wavelength single-molecule fluorescence colocalization methods to follow initiation reactions on individual surface-anchored DNA molecules that contain σ54 promoters. Three distinguishable dye labels enabled us to follow reactions in which RNA polymerase binding, open complex formation, escape into transcription elongation and departure of the σ54 subunit were detected in individual transcription complexes, and the interconversion kinetics for all states were measured. Transcription initiation from this promoter occurs only following a polymerase isomerization that is induced by interaction with the NtrC activator protein in the presence of ATP. However, we observed that with NtrC present the polymerase leaves the promoter faster than the combined rates of initiation plus closed complex departure. Thus, a fraction of activator-mediated polymerase isomerizations displace the polymerase from the promoter without initiating transcript synthesis. This activator-induced abortive isomerization is a non-productive branch off of the initiation pathway and is more common than productive transcription initiation. We speculate that abortive isomerization is a consequence of the large energy input required to disrupt promoter-polymerase interactions prior to promoter escape. Taken together, our results define the full pathway and dynamics of initiation at this activator-dependent promoter and illustrate the power of multi-wavelength single-molecule colocalization methods in the elucidation of complex biological regulatory mechanisms.