Use of saturation mutagenesis to localize probable functional domains in the NahR protein, a LysR-type transcription activator.
Use of saturation mutagenesis to localize probable functional domains in the NahR protein, a LysR-type transcription activator.
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DOI：
10.1016/s0021-9258(19)39671-1
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发表时间：
1990-03
期刊：
影响因子：
--
通讯作者：
M. Schell;P. Brown;S. Raju
中科院分区：
文献类型：
--
作者：
M. Schell;P. Brown;S. Raju
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.
