Abstract
Understanding the pathway and kinetic mechanisms of transcription initiation is essential for quantitative understanding of gene regulation, but initiation is a multistep process, the features of which can be obscured in bulk analysis. We used a multiwavelength single-molecule fluorescence colocalization approach, CoSMoS, to define the initiation pathway at an activator-dependent bacterial σ54 promoter that recapitulates characteristic features of eukaryotic promoters activated by enhancer binding proteins. The experiments kinetically characterize all major steps of the initiation process, revealing heretofore unknown features, including reversible formation of two closed complexes with greatly differing stabilities, multiple attempts for each successful formation of an open complex, and efficient release of σ54 from the polymerase core at the start of transcript synthesis. Open complexes are committed to transcription, suggesting that regulation likely targets earlier steps in the mechanism. CoSMoS is a powerful, generally applicable method to elucidate the mechanisms of transcription and other multistep biochemical processes.
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► Single-molecule approach defines activator-dependent transcription initiation pathway ► Two closed complex intermediates lie on the pathway to σ54-dependent initiation ► Formation of the open transcription complex takes multiple tries ► σ54 departs rapidly as RNA synthesis begins
Multiwavelength optical spectroscopy applied to σ54-dependent transcription reveals a dynamic framework for initiation whereby promoter opening results from an iterative process and σ54 exits rapidly from the polymerase complex upon promoter clearance.