NrpRII mediates contacts between NrpRI and general transcription factors in the archaeon Methanosarcina mazei Gö1

Abstract

We report here on the formation of a complex between the two NrpR homologs present in Methanosarcina mazei Go¨1 and their binding properties to the nifH and glnK 1 promoters. Reciprocal co-chromatography demonstrated that NrpRI forms stable complexes with NrpRII (at an NrpRI : NrpRII molar ratio of 1 : 3), which are not affected by 2-oxoglutarate. Promoter-binding, analyses using DNA-affinity chromatography and electrophoretic gel mobility shift assays, verified that NrpRII is not able to bind to either the nifH promoter or the glnK 1 promoter except when in complex with NrpRI. Specific binding of NrpRI to the nifH and glnK 1 promoters was shown to be highly sensitive to 2-oxoglutarate, regardless of whether only NrpRI, or NrpRI in complex with NrpRII, bound to the promoter. Finally, strong interactions between NrpRII and the general transcription factors TATA-binding proteins (TBP) 1-3 and the general transcription factor TFIIB (TFB) were demonstrated, interactions which are also sensitive to 2-oxoglutarate. On the basis of these findings we propose the following: under nitrogen sufficiency NrpRII binds from solution to either the nifH promoter or the glnK 1 promoter by simultaneously contacting NrpRI and TBP plus TFB, resulting in full repression of transcription; whereas, under nitrogen limitation, increasing 2-oxoglutarate concentrations significantly decrease the binding of NrpRI to the operator as well as the binding of NrpRII to TBP and TFB, ultimately allowing recruitment of RNA polymerase to the promoter.

Towards a Computational Model of a Methane Producing Archaeum

“…However, support for direct regulation of all 83 genes by Nrp regulators was not established, and these connections are instead included in the indirect interaction map. The method of action for repression was shown to be that NrpRI binds the DNA and NrpRII which interacts directly with the TBPs, preventing the RNAP from binding [78]. Importantly, homologs of NrpRI and NrpRII have been identified in M. acetivorans that were differentially expressed under nitrogen limiting versus nitrogen sufficient growth [79], and it is likely that these highly conserved (92–94% identical amino acid sequences) regulators have similar function.…”

Activation of Archaeal Transcription Mediated by Recruitment of Transcription Factor B

“…In this context, it would also be interesting to know whether transcriptional activators exist that are able to promote the recruitment of both TBP and TFB. Interaction with both basal transcription factors was recently described for a repressor involved in the regulation of nitrogen metabolism in Methanosarcina mazei Gö1 (38). Meanwhile, more and more members are being identified for an additional group of archaeal transcriptional regulators that can act as both repressors and activators depending on the location of the binding site, including TrmBL1, Tgr, and SurR (21,22,39).…”

The Emergence of 2-Oxoglutarate as a Master Regulator Metabolite

“…The M. mazei NrpR1 and NrpR2 proteins physically interact and bind as a complex to the nifH and glnK1 promoters in the presence of low 2-OG levels (99). The NrpR2 polypeptide also interacts with the general transcriptional factors (TBP and TFB) in the presence of low 2-OG levels.…”

“…The NrpR2 polypeptide also interacts with the general transcriptional factors (TBP and TFB) in the presence of low 2-OG levels. Hence, the NrpR1-NrpR2 complex binds to operator sequences when the 2-OG concentration is low, through both the NrpR1 DNA-binding HTH motif and interactions between NrpR2 and the general transcriptional factors; this allows repression of nitrogen assimilation genes under nitrogen-sufficient conditions (99).…”