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Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with co-activators to activate transcription. Historically, DNA-binding domains and TADs have been considered as modular units, but recent studies have shown that TADs can influence DNA binding. We biophysically characterized the NFκB p50/RelA heterodimer including the RelA TAD and investigated the influence of the TAD on NFκB-DNA interactions. In solution the RelA TAD is disordered but compact, with helical tendency in two regions that interact with co-activators. The presence of the TAD increased the stoichiometry of NFκB-DNA complexes containing promoter DNA sequences with tandem κB recognition motifs by promoting the binding of NFκB dimers in excess of the number of κB sites. We measured the binding affinity of p50/RelA for DNA containing tandem κB sites and single κB sites. While the presence of the TAD enhanced the binding affinity of p50/RelA for all κB sequences tested, it increased the affinity for non-specific DNA sequences by over 10-fold, leading to an overall decrease in specificity for κB DNA sequences. Our results reveal a novel function of the RelA TAD in promoting binding to non-consensus DNA previously observed by in vivo studies of NFκB-DNA binding in response to strong inflammatory signals.

Sulfate‐reducing bacteria play a key role in the global carbon and sulfur cycles; a role realized through complex competitive or syntrophic metabolic relationships. These relationships are established in response to sulfate levels as well as concentrations of other nutrients such as short chain fatty acids (SCFAs). For some prokaryotes, SCFA production or consumption requires an ATP‐dependent reaction catalyzed by a SCFA kinase. In this study, we have determined the biochemical properties of a butyrate kinase from Desulfovibrio vulgaris str. Hildenborough heterologously expressed in E. coli. Our experiments characterize optimal conditions for activity as well as kinetic parameters for six SCFA substrates and the nucleotide cofactor ATP. These findings have implications on structure and function relationships as well as the potential physiological role of this enzyme. Support or Funding Information The authors would like to thank the Scott and Alice Thomson Fellowship Program for financial support of MB. Further, the authors thank the UW‐Parkside Committee on Research and Creative Activity and the College of Natural and Health Sciences for financial support of this project. Enzymatic Characterization of DvBuk. (A) DvBuk purity assessment was visualized by resolving 10 μg of purified DvBuk on 10% (w/v) polyacrylamide gel and stained with Coomassie Blue. Lane 1: Protein molecular weight markers, Lane 2: recombinant His‐tagged DvBuk. Enzyme activity (%) at various (B) pH and (C) temperature values. (D) Saturation kinetics of DvBuk with butyrate as substrate. (E) Relative activities of DvBuk in the presence of various SCFA substrates. Substrate Preference of DvBuk. (A) Relative activities of DvBuk in the presence of various SCFA substrates (relative activities normalized to observed specific activity in the presence of 200 mM valerate = 9.7 U mg−1). (B) Kinetic constants for DvBuk using various SCFA substrates.

Short and branched chain fatty acid kinases participate in both bacterial anabolic and catabolic processes, including fermentation, through the reversible, ATP-dependent synthesis of acyl phosphates. This study reports biochemical properties of a predicted butyrate kinase from Desulfovibrio vulgaris str. Hildenborough (DvBuk) expressed heterologously and purified from Escherichia coli. Gel filtration chromatography indicates purified DvBuk is active as a dimer. The optimum temperature and pH for DvBuk activity is 44°C and 7.5, respectively. The enzyme displays enhanced thermal stability in the presence of substrates as observed for similar enzymes. Measurement of kcat and KM for various substrates reveals DvBuk exhibits the highest catalytic efficiencies for butyrate, valerate and isobutyrate. In particular, these measurements reveal this enzyme's apparent high affinity for C4 fatty acids relative to other butyrate kinases. These results have implications on structure and function relationships within the ASKHA superfamily of phosphotransferases, particularly regarding the acyl binding pocket, as well as potential physiological roles for this enzyme in Desulfovibrio vulgaris str. Hildenborough.