TRANSLATIONAL ACTIVATION BY AN NTRC ENHANCER-BINDING PROTEIN

The Rhodobacter capsulatus NtrC protein is a bacterial enhancer-bindin g protein that activates the transcription of at least five genes by a mechanism that does not require the RpoN RNA polymerase sigma factor. The nifR3-ntrB-ntrC operon in R. capsulatus codes for the nitrogen-se nsing two component regulators NtrB and NtrC, as well as for NifR3, a protein of unknown function that is highly conserved in both prokaryot es and eukaryotes. Evidence of a unique translational control of NifR3 mediated directly by the NtrC enhancer-binding protein is reported. T he nifR3-ntrB-ntrC operon is expressed from a single promoter upstream of nifR3 with the levels of transcript equivalent in wild-type and nt rC mutants under nitrogen-limited or nitrogen-sufficient conditions. L acZ reporter analyses of this operon and immunological quantitation of NifR3 and NtrC demonstrate that, unlike NtrC levels which remain cons tant, production of NifR3 is at least ten to 40-fold reduced in NtrC(- ) strains. NifR3 is increased at least fivefold upon nitrogen limitati on whereas NtrC production is constitutive. Surprisingly, the purified NtrC protein binds cooperatively to the niJR3 promoter region in vitr o at two sets of tandem binding sites centered at +1 and -81 nucleotid es relative to the transcriptional start site. Deletion analysis demon strates that the upstream tandem sites are essential for nitrogen and NtrC-dependent production of NifR3 in vivo, but are not necessary for nifR3 transcription. These experiments indicate that NtrC stimulates t he translation of the NifR3 messenger RNA while tethered to the promot er DNA. This is in contrast to five other promoters (nifA1, nifA2, gln B, mopA and anfA) in X. capsulatus which are transcriptionally activat ed by NtrC bound to one set of tandem binding sites that are centered greater than 100 bp upstream of the transcriptional start site. (C) 19 98 Academic Press Limited.