Repressor Forms of the Enhancer-binding Protein NtrC: Some Fail in Coupling ATP Hydrolysis to Open Complex Formation by σ54-Holoenzyme

A conserved region in the σ54‐dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation

“…In general, the mutant forms of DctD (⌬1-142) with amino acid substitutions in the GAFTGA motif retained ATPase activity, similar to that reported previously for mutant forms of S. typhimurium NtrC with amino acid substitutions in this same region (Weiss et al, 1991;North et al, 1996). Taken together with the cross-linking data, we postulate that the GAFTGA motif functions to couple energy release from ATP hydrolysis to open complex formation rather than binding the activator to 54 -holoenzyme.…”

“…Substitution of the equivalent amino acid residue in S. typhimurium NtrC, Glu-208, with Gln resulted in loss of transcriptional activation. Like DctD (⌬1-142, E213V) , the ATPase activity of NtrC E208Q was significantly reduced, but it was sufficiently high that the failure of this protein to activate transcription could not be accounted for solely by the defect in ATPase activity (North et al, 1996). DctD (⌬1-142, E213V) could be cross-linked efficiently to both 54 and the ␤-subunit.…”

“…While mutant forms of 54 -dependent activators that fail to catalyse open complex formation have been reported previously (Weiss et al, 1991;North et al, 1996;Perez-Martin and de Lorenzo, 1996a), none of these mutant proteins have been shown to be specifically defective in contacting 54 -holoenzyme. To understand further the molecular mechanisms of activation with 54 -holoenzyme, we sought to identify amino acid residues in DctD that are involved in protein-protein interactions with 54 -holoenzyme.…”

“…1). Secondly, amino acid substitutions in the C3 region of the 54 -dependent activator, Salmonella typhimurium NtrC, interfere with transcriptional activation but do not affect DNA binding or ATPase activities of the protein (North et al, 1996). These mutant proteins are believed to be defective in either protein-protein interactions with 54 -holoenzyme or in coupling energy from ATP hydrolysis to the formation of open complexes.…”

Genetic evidence of separate repressor and activator activities of the XylR regulator of the TOL plasmid, pWWO, of Pseudomonas putida

“…A third mutant not previously described, named A31 1V (encoded by plasmid pVTR4), was also employed. This variant reproduces in XylR one mutation described in NtrC (NtrCA2'6v; North et a/., 1996) that is believed to affect its ability to catalyse open-complex formation while retaining the DNA-binding-and ATP-hydrolysis activities of the wild-type protein. Plasmids encoding repress Pr activity, at least partially, suggests (although it does not prove) that occupation of as little as one of the two binding sites in Ps (Perez-Martin and de Lorenzo, 1996d) may be sufficient for the downregulation effect.…”