Abstract
Numerous in vitro studies have yielded a refined picture of the structural and molecular associations between Cyclic-AMP receptor protein (Crp), the DNA motif, and RNA polymerase (RNAP) holoenzyme. In this study, high-resolution ChIP-exonuclease (ChIP-exo) was applied to study Crp binding in vivo and at genome-scale. Surprisingly, Crp was found to provide little to no protection of the DNA motif under activating conditions. Instead, Crp demonstrated binding patterns that closely resembled those generated by σ70. The binding patterns of both Crp and σ70 are indicative of RNAP holoenzyme DNA footprinting profiles associated with stages during transcription initiation that occur post-recruitment. This is marked by a pronounced advancement of the template strand footprint profile to the +20 position relative to the transcription start site and a multimodal distribution on the nontemplate strand. This trend was also observed in the familial transcription factor, Fnr, but full protection of the motif was seen in the repressor ArcA. Given the time-scale of ChIP studies and that the rate-limiting step in transcription initiation is typically post recruitment, we propose a hypothesis where Crp is absent from the DNA motif but remains associated with RNAP holoenzyme post-recruitment during transcription initiation. The release of Crp from the DNA motif may be a result of energetic changes that occur as RNAP holoenzyme traverses the various stable intermediates towards elongation complex formation.
Highlights
Cyclic-AMP receptor protein (Crp) is the most thoroughly characterized transcription factor from a structural and mechanistic standpoint [1,2,3]
Strand oriented peak distributions reveal stable intermediates in transcription initiation The σ70 Chromatin immunoprecipitation (ChIP)-exo peak distribution provides the bounds of protected DNA regions on the template and nontemplate strand
Venn diagram showing pairwise comparison of peaks regions detected for ΔAr1, ΔAr2, and ΔAr1ΔAr2 with wild type Crp
Summary
Crp (cAMP receptor protein; known as CAP, catabolite activator protein) is the most thoroughly characterized transcription factor from a structural and mechanistic standpoint [1,2,3]. This classic approach utilizes the protection from nuclease digestion provided by proteins bound to DNA to produce a highly precise map of the binding site [20] This method has been extensively applied to study the mechanics and kinetics of transcription initiation events [21,22,23]. Class III promoters involve two Crp molecules and a second transcription factor that often represses the activating action of Crp. Footprinting studies under highly controlled and stabilizing conditions have shown that the Crp motif sequence is protected when in complex with Crp and RNAP holoenzyme [29,30,31,32]. This enables precise identification of binding events by combining DNA footprinting with ChIP This method has been applied to the study of eukaryotic pre-initiation complexes, which is typical comprised of RNAP II and no less than six additional general transcription factors [34]. Genetic perturbations to Crp/RNAP interactions were introduced and the affects of these mutations were characterized using ChIP-exo
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