Mutations in the RNA polymerase recognition sequence of theKlebsiella pneumoniae nifHpromoter permitting transcriptional activation in the absence of NifA binding to upstream activator sequences

Abstract

Positive control of the wild-type Klebsiella pneumoniae nifH promoter by the NifA protein requires that NifA is bound at the upstream activator sequence (UAS). By introducing base substitutions at -15 to -17 in the RNA polymerase recognition sequence of the nifH promoter, positive control by a form of NifA unable to bind to the UAS was greatly increased when compared to the wild-type promoter. Transcriptional activation still required the rpoN encoded sigma factor and was initiated at the same nucleotide as in the wild-type promoter. Mutations at -15 to -17 suppressed the requirement that the UAS should be located on the correct face of the DNA helix with respect to the RNA polymerase recognition sequence in order that titration of NifA and efficient activation occur. This result supports the suggestion that upstream bound NifA interacts with the RNA polymerase-RpoN complex. To examine the minimal carboxy terminal sequences required for the positive control function of NifA a series of carboxy terminal deletions were constructed. Efficient positive control at a UAS-independent promoter was only observed in deletions which did not extend beyond the proposed boundary separating the carboxy terminal NifA DNA-binding domain from its central domain.