The function of isolated domains and chimaeric proteins constructed from the transcriptional activators NifA and NtrC of Klebsiella pneumoniae.
A model for the domain structure of sigma 54-dependent transcriptional activators, based on sequence data, has been tested by examining the function of truncated and chimaeric proteins. Removal of the N-terminal domain of NtrC abolishes transcriptional activation, indicating that this domain is positively required for activator function. Over-expression of this domain as a separate peptide appears to titrate out the phosphorylating activity of NtrB. Removal of the N-terminal domain of NifA reduces activation 3-4-fold. The residual activity is particularly sensitive to inhibition by NifL, suggesting that the role of the N-terminal domain is to block the action of NifL in derepressing conditions. The C-terminal domain of NtrC showed repressor activity when expressed as a separate peptide. This domain is necessary for activator function even when NtrC binding sites are deleted from promoters. A point mutation in the ATP-binding motif of the NtrC central domain, Ser169 to Ala, also abolished activator function. Exchanging the N-terminal domains of Klebsiella pneumoniae NtrC, NifA and Escherichia coli OmpR, did not produce any hybrid activity, suggesting that N-terminal domains in the native proteins specifically recognize the rest of the molecule.
Drummond
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MH
AFRC Institute of Plant Science Research, University of Sussex, Brighton, UK.
Contreras
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A
Mitchenall
L A
LA
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D007711:Klebsiella pneumoniae / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D051922:PII Nitrogen Regulatory Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061338:NifA protein, Bacteria; D051922:PII Nitrogen Regulatory Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli