Amino acid substitutions in the CytR repressor which alter its capacity to regulate gene expression

Abstract

In Escherichia coli, transport and catabolism of nucleosides require expression of the genes composing the CytR regulon. Transcription initiation of cistrons in this gene family is activated by cyclic AMP-catabolite activator protein (cAMP-CAP), repressed by the CytR protein, and induced by cytidine. A random proofreading mutagenesis procedure and a genetic screen using udp-lac fusions have allowed the identification of distinct regions of the 341-amino-acid CytR polypeptide that are critical for repression of gene expression and response to induction. Determination of the abilit of various CytR mutants to control gene expression in vivo indicated that the intrinsic affinity of the CytR protein for operator DNA is gene specific and that efficient repression of transcription by wild-type CytR is dependent on the interaction of CytR with cAMP-CAP. CytR mutants that were cytidine induction defective (CID) were characterized; these mutant proteins had only Asp-281 replaced. Data obtained with cytRAM149, a dominant negative allele, indicated that the native CytR repressor is an oligomeric protein. Representative cytR mutations were combined with cytRAM149, and the resulting hybrid repressors were tested for transdominance in a CytR+ E. coli strain. Amino acid substitutions A209E and C289Y suppressed the transdominance of CytRAM149, suggesting that these replacements alter the normal protein contacts involved in repressor subunit-subunit association. In contrast, amino acid substitutions located in the N-terminal portion of the CytR protein had no effect on the transdominance of CytRAM149. The results from this study suggest that the CytR repressor is an oligomeric, allosteric protein in which conformational changes are required for repression and derepression.

Characterization of cytR mutations that influence oligomerization of mutant repressor subunits

“…CytRAM149 is a totally inactive, mutant repressor that has been shown to be transdominant when synthesized from a multicopy plasmid in a strain that expresses the wild-type protein from the chromosome. The transdominance of CytRAM149 is thought to result from formation of inactive, heterooligomeric repressors composed of wild-type and CytRAM149 subunits, thus suggesting that the overall structures for both proteins are comparable (1). Accordingly, any second-site mutation that encodes an amino acid substitution found to suppress the transdominant phenotype of CytRAM149 should either affect normal repressor subunit-subunit interaction or the proper folding of the mutant protein.…”

“…In every case except for the clone expressing CytRL311(Am) (discussed below), the CDase and UDP specific activities were equivalent to those recorded for the CytR-control strain. Since we have previously shown (1) that the CytR C terminus is essential for repressor activity, the partial repression of cdd and udp expression found with SS6018 expressing CytRL311(Am) is likely due to endogenous suppression of the amber translation termination codon. This conclusion is supported by the detection of both full-length and truncated CytR proteins in cell extracts of SS6018 expressing repressor from the cytRL311(Am) allele (Fig.…”

“…To confirm that the recessive phenotype of SAD mutants resulted from their inabilities to oligomerize, we constructed a set of chimeric genes which contained both the dominant negative cytRAM149 mutation (1) (Table 4). In contrast, coexpression of each hybrid CytR repressor with the wildtype protein had no effect on the ability of wild-type CytR to regulate cdd and udp transcription.…”

“…The abilities of CytRAM149 and CytRA321-341 mutant proteins to interact with and thus titrate functional wild-type subunits provides us with a tool for assessing the effects of secondary mutations on CytR subunit oligomerization (1). In this genetic test, termed the suppression of transdominance assay (Fig.…”

“…Electrophoresis of cell extract protein samples was as described above, except that 8 ,ug of extract protein was analyzed per gel lane. Following electrophoresis, transfer of the proteins separated by SDS-polyacrylamide gel electrophoresis to Immobilon membrane and detection of the CytR protein were carried out as described previously (1).…”