Dataset Information

Mutational analysis of intervening sequences connecting the binding sites for integration host factor, PepA, PurR, and RNA polymerase in the control region of the Escherichia coli carAB operon, encoding carbamoylphosphate synthase.

ABSTRACT: Transcription of the carAB operon encoding the unique carbamoylphosphate synthase of Escherichia coli reflects the dual function of carbamoylphosphate in the biosynthesis of arginine and pyrimidine nucleotides. The tandem pair of promoters is regulated by various mechanisms depending on the needs of both pathways and the maintenance of a pyrimidine/purine nucleotide balance. Here we focus on the linker regions that impose the distribution of target sites for DNA-binding proteins involved in pyrimidine- and purine-specific repression of the upstream promoter P1. We introduced deletions and insertions, and combinations thereof, in four linkers connecting the binding sites for integration host factor (IHF), PepA, PurR, and RNA polymerase and studied the importance of phasing and spacing of the targets and the importance of the nucleotide sequence of the linkers. The two PepA binding sites must be properly aligned and separated with respect to each other and to the promoter for both pyrimidine- and purine-mediated repression. Similarly, the phasing and spacing of the IHF and PEPA2 sites are strictly constrained but only for pyrimidine-specific repression. The IHF target is even dispensable for purine-mediated regulation. Thus, a correct localization of PepA within the higher-order nucleoprotein complex is a prerequisite for the establishment of pyrimidine-mediated repression and for the coupling between purine- and pyrimidine-dependent regulation. Our data also suggest the existence of a novel cis-acting pyrimidine-specific regulatory target located around position -60. Finally, the analysis of a P1 derivative devoid of its control region has led to a reappraisal of the effect of excess adenine on P1 and has revealed that P1 has no need for a UP element.

SUBMITTER: Devroede N

PROVIDER: S-EPMC1447446 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Mutational analysis of intervening sequences connecting the binding sites for integration host factor, PepA, PurR, and RNA polymerase in the control region of the Escherichia coli carAB operon, encoding carbamoylphosphate synthase.

Devroede Neel N Huysveld Nadine N Charlier Daniel D

Journal of bacteriology 20060501 9

Transcription of the carAB operon encoding the unique carbamoylphosphate synthase of Escherichia coli reflects the dual function of carbamoylphosphate in the biosynthesis of arginine and pyrimidine nucleotides. The tandem pair of promoters is regulated by various mechanisms depending on the needs of both pathways and the maintenance of a pyrimidine/purine nucleotide balance. Here we focus on the linker regions that impose the distribution of target sites for DNA-binding proteins involved in pyri ...[more]

PMID: 16621816

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Project description:Homologue function can be differentiated by changing residues that affect binding sites or long-range interactions. LacI and PurR are two proteins that represent the LacI/GalR family (>500 members) of bacterial transcription regulators. All members have distinct DNA-binding and regulatory domains linked by approximately 18 amino acids. Each homologue has specificity for different DNA and regulatory effector ligands; LacI and PurR also exhibit differences in allosteric communication between DNA and effector binding sites. A comparative study of LacI and PurR suggested that alterations in the interface between the regulatory domain and linker are important for differentiating their functions. Four residues (equivalent to LacI positions 48, 55, 58, and 61) appear particularly important for creating a unique interface and were predicted to be necessary for allosteric regulation. However, nearby residues in the linker interact with DNA ligand. Thus, differences observed in interactions between linker and regulatory domain may be the cause of altered function or an effect of the two proteins binding different DNA ligands. To separate these possibilities, we created a chimeric protein with the LacI DNA-binding domain/linker and the PurR regulatory domain (LLhP). If the interface requires homologue-specific interactions in order to propagate the signal from effector binding, then LLhP repression should not be allosterically regulated by effector binding. Experiments show that LLhP is capable of repression from lacO1 and, contrary to expectation, allosteric response is intact. Further, restoring the potential for PurR-like interactions via substitutions in the LLhP linker tends to diminish repression. These effects are especially pronounced for residues 58 and 61. Clearly, binding affinity of LLhP for the lacO1 DNA site is sensitive to long-range changes in the linker. This result also raises the possibility that mutations at positions 58 and 61 co-evolved with changes in the DNA-binding site. In addition, repression measured in the absence and presence of effector ligand shows that allosteric response increases for several LLhP variants with substitutions at positions 48 and 55. Thus, while side chain variation at these sites does not generally dictate the presence or absence of allostery, the nature of the amino acid can modulate the response to effector.

Dataset Information

Mutational analysis of intervening sequences connecting the binding sites for integration host factor, PepA, PurR, and RNA polymerase in the control region of the Escherichia coli carAB operon, encoding carbamoylphosphate synthase.

Publications

Mutational analysis of intervening sequences connecting the binding sites for integration host factor, PepA, PurR, and RNA polymerase in the control region of the Escherichia coli carAB operon, encoding carbamoylphosphate synthase.

Similar Datasets

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