Kinetically controlled competition between NusA and σ70 yields functionally diverse transcription elongation complexes
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PROVIDER: PRJNA435408 | ENA |
REPOSITORIES: ENA
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Project description:In all organisms, the multi-subunit RNA polymerases (RNAPs) that synthesize messenger RNAs bind multiple accessory proteins to regulate transcript elongation rate, transcriptional pausing, and termination. However, the dynamics of regulatory protein association/dissociation and how different regulators influence one another’s function is unclear. We used single-molecule multi-wavelength fluorescence colocalization techniques to directly observe the dynamics of the elongation regulators NusA and σ70 with E. coli RNAP in vitro. Contrary to previous proposals, NusA was observed to repeatedly bind to and release from elongation complexes (ECs) during synthesis of a single RNA. However, elongation complexes that retained bound σ70 did not bind NusA and the EC-bound σ70 was largely retained even in the presence of physiological (µM) concentrations of competing elongation factors NusA and/or NusG. Factor occupancy of elongation complexes was non-equilibrium, with substantial amounts of σ70ECs even in the absence of free σ70, because composition was controlled by slow σ70 dissociation from σ70ECs. The experiments show that at steady state the same gene is transcribed by two distinct types of elongation complexes that are (ECs) or are not (σ70ECs) capable of binding NusA. Consistent with the known regulatory effects of NusA, the two types are observed to have different elongation rates, pausing dynamics, and efficiency of termination at an intrinsic terminator. During cellular transcription, these non-equilibrium populations of ECs are predicted to cause selective EC loss at intergenic transcriptional attenuators, traffic jams and altered pausing. This is an example of a potentially general “two-body” mechanism in which a functionally silent protein fine-tunes gene expression by modulating a second, functionally consequential regulator.
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