Transcriptional activation of the nitrogenase promoter in vitro: adenosine nucleotides are required for inhibition of NIFA activity by NIFL

Abstract

The enhancer-binding protein NIFA is required for transcriptional activation of nif promoters by the alternative holoenzyme form of RNA polymerase, which contains the sigma factor 54 ( N ). NIFA hydrolyzes nucleoside triphosphates to catalyze the isomerization of closed promoter complexes to transcriptionally competent open complexes. The activity of NIFA is antagonized by the regulatory protein NIFL in response to oxygen and fixed nitrogen in vivo. We have investigated the requirement for nucleotides in the formation and stability of open promoter complexes by NIFA and inhibition of its activity by NIFL at the Klebsiella pneumoniae nifH promoter. Open complexes formed by 54 -containing RNA polymerase are considerably more stable to heparin challenge in the presence of GTP than in the presence of ATP. This differential stability is most probably a consequence of GTP being the initiating nucleotide at this promoter. Adenosine nucleosides are specifically required for Azotobacter vinelandii NIFL to inhibit open complex formation by native NIFA, and the nucleoside triphosphatase activity of NIFA is strongly inhibited by NIFL under these conditions. We propose a model in which NIFL modulates the activity of NIFA via an adenosine nucleotide switch.A distinct mechanism of transcriptional activation is observed among the family of prokaryotic enhancer-binding proteins which interact with the holoenzyme form of RNA polymerase containing the alternative sigma factor 54 (E 54 ) (19,26). The nitrogen fixation regulatory protein NIFA is a member of this family which binds to upstream activator sequences (UAS) and catalyzes the isomerization of closed promoter complexes to the open complex in a reaction which requires hydrolysis of a nucleoside triphosphate (25). Productive interactions between NIFA and E 54 are enabled by DNA loop formation, which is facilitated by the binding of integration host factor (IHF) (18,30). The amino acid sequence of NIFA conforms to the three-domain model for the structure of pneumoniae NIFA activates transcription in the absence of specific DNA binding and possesses nucleoside triphosphatase activity (6). In contrast to its K. pneumoniae counterpart, the native Azotobacter vinelandii NIFA protein has been purified in a soluble form, and its properties with respect to DNA binding and catalysis of open complex formation have been characterized in vitro (2).In both K. pneumoniae and A. vinelandii, the activity of NIFA is controlled by a second regulatory protein, NIFL, in response to the environmental effectors oxygen and fixed nitrogen (7,22). Although NIFL proteins show homology in their C-terminal domains to the histidine protein kinase family of two-component regulatory proteins (14), NIFL and NIFA appear to interact at stoichiometric levels (5, 15), and phosphotransfer between the two proteins has not been detected in vitro (2, 21). Moreover, although A. vinelandii NIFL shows greater homology to the canonical histidine protein kinases than does K. pneumoniae NIFL and contains a conserved...

The Amino-terminal GAF Domain of Azotobacter vinelandii NifA Binds 2-Oxoglutarate to Resist Inhibition by NifL under Nitrogen-limiting Conditions

“…The inhibitory activity of NifL is stimulated by adenosine nucleotides to promote formation of the NifL-NifA complex (5)(6)(7). This protein-protein interaction sequesters NifA and inhibits ATP hydrolysis by the activator, thus preventing productive interactions with 54 -RNA polymerase and inhibiting transcriptional activation at nif promoters.…”

“…Lanes 4,7,10,13,16,19, and 22 contained NifA, ATP, and 2-oxoglutarate incubated for t ϭ 0, 2, 6, 10, 30, 60, and 100 min, respectively. Lanes 5,8,11,14,17,20, and 23 contained NifA, ATP, and 3-oxoglutarate incubated for t ϭ 0, 2, 6, 10, 30, 60, and 100 min, respectively. Lane 2 shows purified NifA as a marker.…”

A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii

“…NifA-promoted catalysis of openpromoter complexes by 54 -RNA polymerase was used to assay NifA activity and its inhibition by NifL as described (16,18).…”

“…1). Unlike the HPKs, the GHKL domain of NifL does not exhibit ATP hydrolysis or transphosphorylation activity, but the binding of ADP to this domain strongly stimulates the inhibitory activity of NifL and the stability of the NifL-NifA complex (8,15,16). Also, the GHKL domain is involved in sensing the nitrogen status because it is the site of interaction with the signal transduction protein GlnK (17)(18)(19), the PII-like protein of A. vinelandii (20)(21)(22)(23).…”

Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae

“…However, NifL and NifA are produced in roughly similar amounts, and coimmunoprecipitation tests suggested that they may form a complex (12). Also, total inhibition of NifA function in vitro requires a molar excess of NifL, either purified or reconstituted from inclusion bodies (9,20), so the action of NifL on NifA appears to be mediated by direct protein-protein interactions. If this is the case, a correct NifLNifA stoichiometry must be essential for regulation of nitrogen fixation, since synthesis of an excess of NifA could lead to a substantial production of many nif gene products under conditions in which they are unnecessary or useless.…”