Structure, function, and tethering of DNA‐binding domains in σ⁵⁴ transcriptional activators

Abstract

We compare the structure, activity and linkage of DNA binding domains from σ54 transcriptional activators, and discuss how the properties of the DNA binding domains and the linker to the neighboring domain are affected by the overall properties and requirements of the full proteins. These transcriptional activators bind upstream of specific promoters that utilize σ54-polymerase. Upon receiving a signal the activators assemble into hexamers, which then, through ATP hydrolysis, drive a conformational change in polymerase that enables transcription initiation. We present structures of the DNA-binding domains of activators NtrC1 and Nlh2 from the thermophile A. aeolicus. The structures of these domains, and their relationship to other sparts of the activators are discussed. These structures are compared with previously determined structures of the DNA-binding domains of NtrC4, NtrC, ZraR, and FIS. The N-terminal linkers that connect the DNA-binding domains to the central domains in NtrC1 and Nlh2 were studied and found to be unstructured. Additionally, a crystal structure of full-length NtrC1 was solved, but density for the DNA-binding domains was extremely weak, further indicating that the linker between ATPase and DNA binding domains functions as a flexible tether. Flexible linking of ATPase and DNA binding domains is likely necessary to allow assembly of the active hexameric ATPase ring. The comparison of this set of activators also shows clearly that strong dimerization of the DNA binding domain only occurs when other domains do not dimerize strongly.

DNA Recognition by a σ54 Transcriptional Activator from Aquifex aeolicus

“…In NtrC residues identified as important for DNA binding through mutagenesis (R456, N457, R461) 38 are in the recogniton helix, and are predicted to point into the groove, the difference in spacing of the helices 39 leaves unclear what bending is induced by NtrC and which residues are responsible. Localized bending may affect the cooperativity of binding activators at adjacent sites, and hence may affect potency of activator sites.…”

Nucleotide-induced asymmetry within ATPase activator ring drives σ54–RNAP interaction and ATP hydrolysis

“…Mass spectroscopy data showed that hexamer is favored over heptamer in fulllength constructs of NtrC4, an EBP related to NtrC1 (Batchelor et al 2009). The receiver domain forms stable homodimers, and the C-terminal DNA-binding domain directs assembly of four protomers in a tandem array on ''enhancers'' (Doucleff et al 2005;Vidangos et al 2013). However, this potential artifact underscores the stabilization of split hexamer rings of EBP and does not diminish the evidence for g-phosphate-directed asymmetry in this structural state.…”

Kinetics of transcription initiation directed by multiplecis-regulatory elements on theglnAp2promoter

“…glnAp2 transcription is activated by NtrC in response to nitrogen limitation (29–33). NtrC molecules are dimeric in their inactive state.…”

“…σ 54 RNAP binds to the −24–−12 region at one face of the double helix (11). NtrC hexamers catalyze σ 54 RNAP via DNA looping; the catalysis reaction takes place at approximately −12 region and the edge of the central pore of NtrC hexamer (10,11,29,38). Such a regulatory mode—activators at remote enhancers direct transcription initiation though DNA looping—is similar to that in eukaryotes.…”

“…The notion that transcription initiation is activated by an NtrC hexamer spanning the two enhancers fails to explain why the low-affinity sites can promote transcriptional output (35,41). Structurally, the binding of NtrC to the low-affinity sites enhances the bending rigidity of DNA, rather than significantly bend DNA as the integration host factor (7), and thus prevents the hexamers at enhancers from contacting the holoenzyme (29,36,42). An alternative postulation was that NtrC dimers could constitute a huge octamer at the five sites to activate transcription (22) (Figure 1D); but this is inconsistent with the structural basis of activation—the holoenzyme is catalyzed at the edge of the central pore of NtrC hexamer (10,11,29,38).…”