The bacterial cell cycle regulator GcrA is a σ⁷⁰ cofactor that drives gene expression from a subset of methylated promoters

Abstract

Cell cycle progression in most organisms requires tightly regulated programs of gene expression. The transcription factors involved typically stimulate gene expression by binding specific DNA sequences in promoters and recruiting RNA polymerase. Here, we found that the essential cell cycle regulator GcrA in Caulobacter crescentus activates the transcription of target genes in a fundamentally different manner. GcrA forms a stable complex with RNA polymerase and localizes to almost all active σ 70 -dependent promoters in vivo but activates transcription primarily at promoters harboring certain DNA methylation sites. Whereas most transcription factors that contact σ 70 interact with domain 4, GcrA interfaces with domain 2, the region that binds the −10 element during strand separation. Using kinetic analyses and a reconstituted in vitro transcription assay, we demonstrated that GcrA can stabilize RNA polymerase binding and directly stimulate open complex formation to activate transcription. Guided by these studies, we identified a regulon of ∼200 genes, providing new insight into the essential functions of GcrA. Collectively, our work reveals a new mechanism for transcriptional regulation, and we discuss the potential benefits of activating transcription by promoting RNA polymerase isomerization rather than recruitment exclusively.

Cell cycle progression inCaulobacterrequires a nucleoid-associated protein with high AT sequence recognition

“…3D). To determine whether GapR associates with promoters that are normally active during growth, we measured overlap between the GapR ChIP-seq profile with that of the housekeeping sigma factor in Caulobacter (σ 73 , i.e., RpoD) trapped on DNA in initiation complexes with target promoters as a consequence of rifampicin treatment (33). We observed a correlation between GapR and RpoD occupancy, with 80.4% of the top 500 GapR peaks overlapping an RpoD peak (Fig.…”

“…12; GcrA ChIP-seq data were obtained from ref. 33 and peaks were called using the same workflow used for GapR/CtrA (see SI Materials and Methods for details). (C) Network diagram of intergenic regions (gray, purple, and yellow nodes) associated with different proteins (red, green, blue, and orange nodes).…”

An intracellular compass spatially coordinates cell cycle modules in Caulobacter crescentus

“…Transcription of ftsZ largely occurs in S-phase, concurrent with the initiation of DNA replication (Figure 1a), as a result of combinatorial transcriptional control by the global regulators DnaA, GcrA, and CtrA [3 • ,4]. Additionally, deletion of the methyltransferase ccrM dramatically reduces ftsZ expression [7], likely by decreasing GcrA-dependent transcription [8 •• ]. Notably, a ccrM deletion can be rescued by mutations in the phosphoenolpyruvate-carbohydrate phosphotransfer system (PTS) that upregulates ftsZ transcription, suggestive of additional layers of nutrient-sensing transcriptional regulation [9,10].…”

“…Notably, a ccrM deletion can be rescued by mutations in the phosphoenolpyruvate-carbohydrate phosphotransfer system (PTS) that upregulates ftsZ transcription, suggestive of additional layers of nutrient-sensing transcriptional regulation [9,10]. Indeed, several studies have linked expression levels of GcrA-regulated transcription to levels of the alarmone (p)ppGpp [8 •• ,10,11]. …”

The impact of DNA methylation in Alphaproteobacteria

“…In addition, the phenotypes of ∆ gcrA and ∆ ccrM mutants displayed striking similarities, being incapable of dividing under fast‐growing conditions (Gonzalez and Collier, ; Murray et al ., ). GcrA is a cell cycle‐regulated transcriptional regulator that controls the expression of hundreds of genes when it is present in S‐phase cells (Holtzendorff et al ., ; Fioravanti et al ., ; Haakonsen et al ., ) and it is often co‐conserved with CcrM in Alphaproteobacteria (Brilli et al ., ; Murray et al ., ). Its mechanism of action remained unclear for over a decade after its discovery, in large part due to its unique domains that did not resemble canonical DNA binding domains.…”