The bacterial RNA polymerase-associated CarD protein couples promoter activity to DNA supercoiling

Abstract

Abstract RNA polymerase (RNAP) must unwind duplex DNA prior to transcription initiation. In bacteria, unwinding starts at a DNA motif called the promoter −10 element. Specifically, non-template strand bases interact with RNAP to trigger the process. A protein called CarD can support −10 element opening in many microbes. Whilst this most often activates transcription, CarD may repress if the DNA open complex is too stable. For Rhodobacter sphaeroides , a purple photoheterotrophic alpha-proteobacterium, CarD is particularly important as many −10 elements have a key DNA sequence defect. Here, we use genomic tools to map transcription initiation, binding of RNAP, CarD interactions, and DNA topology, globally in R. sphaeroides . We show an association between CarD-controlled transcription, and negative DNA supercoiling, which alters global gene expression if perturbed. Using biochemical tools, we show that promoter co-regulation by CarD and supercoiling results from the −10 element defect inherent to R. sphaeroides promoters. If this flaw is corrected, regulation by CarD and supercoiling is disrupted. As supercoiling dissipates during stress, we suggest CarD couples housekeeping transcription to the environment via DNA topology.