Use of saturation mutagenesis to localize probable functional domains in the NahR protein, a LysR-type transcription activator.
The NahR protein of the Pseudomonas naphthalene degradation plasmid NAH7 encodes a 300-residue transcription activator which is very similar to the NodD transcription activator of Rhizobium and other proteins in the LysR activator family. NahR binds to conserved sequences upstream (nucleotides -80 to -47) of the nah and sal promoters and activates transcription of genes for naphthalene catabolism in response to the inducer salicylate. Transformation of an Escherichia coli gal deletion strain (containing a sal promoter-galK fusion plasmid) with hydroxylamine-treated nahR DNA and selection on galactose/salicylate plates allowed isolation of 30 unique activation-deficient nahR alleles which fell into two classes: class I, defective in both activation and specific binding to the NahR activation site of the sal promoter; and class II, defective in activation, but with wild-type DNA binding activity. DNA sequence analysis showed that the amino acid substitutions eliminating DNA binding activity were mostly clustered in an NH2-terminal helix-turn-helix motif (residues 23-45) or a COOH-terminal domain (residues 239-291). Similar analysis of class II mutants identified a domain (residues 126-206) possibly involved in inducer binding and/or transcription activation functions. The partial trans-dominance of many mutant alleles and the size of NahR-specific DNA binding activity measured by gel filtration suggest that the active NahR protein may be a tetramer.
Schell
M A
MA
Department of Microbiology, University of Georgia, Athens 30602.
Brown
P H
PH
Raju
S
S
D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002872:Chromosome Deletion; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009281:Naphthalenes / Q000378:metabolism; D009876:Operon; D010957:Plasmids; D011549:Pseudomonas / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006898:Hydroxylamines; C063173:NahR protein, Bacteria; D009281:Naphthalenes; D014157:Transcription Factors; C031721:naphthalene; D019811:Hydroxylamine