Characterization of cytR mutations that influence oligomerization of mutant repressor subunits.
In Escherichia coli, the transport and catabolism of nucleosides require expression of the genes composing the CytR regulon. The role of the CytR repressor in transcriptional regulation has been examined through a study of mutant CytR proteins. Two important and interrelated CytR mutants are encoded by cytR delta M149, a dominant negative allele, and cytRC289R. Studies with CytR delta M149 indicated that the native, repression-competent CytR protein is multimeric while the CytR amino acid substitution C-289-->R has been proposed to affect subunit oligomerization on the basis of its ability to suppress the transdominance of CytR delta M149. The present study identifies other CytR amino acid residues proximal to Cys-289 that may also participate in normal subunit oligomerization. Mutations in these CytR residues, cytRA307P, cytRM308R, and cytRL309P, encoded inactive repressors in a CytR- background and, when combined with cytR delta M149, yielded hybrid repressors that were recessive in a CytR+ genetic background. Because the stability and solubility observed for the new, mutant CytR proteins and the wild-type CytR protein were indistinguishable, these residue replacements, like the C-289-->R substitution, are envisaged as being located at the subunit interface and thus suppress the CytR delta M149 transdominance by blocking efficient and stable assembly of wild-type and hybrid CytR subunits. The assignment of CytR amino acids to a protein region involved in subunit association is also consistent with the observations that these CytR amino acids are roughly colinear with regions of the LacI repressor that influence monomer-dimer association and would be surface located by alignment to the E. coli galactose-binding protein crystal structure.
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Division of Molecular Genetics and Microbiology, Wellcome Research Laboratory, Research Triangle Park, North Carolina 27709.
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D005798:Genes, Bacterial / Q000235:genetics* / Q000502:physiology; D005809:Genes, Regulator / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics* / Q000502:physiology; D012097:Repressor Proteins / Q000235:genetics* / Q000502:physiology
D001426:Bacterial Proteins; C048425:CytR protein, E coli; D029968:Escherichia coli Proteins; D012097:Repressor Proteins