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Crystal structure of KorA bound to operator DNA: insight into repressor cooperation in RP4 gene regulation.

ABSTRACT: KorA is a global repressor in RP4 which regulates cooperatively the expression of plasmid genes whose products are involved in replication, conjugative transfer and stable inheritance. The structure of KorA bound to an 18-bp DNA duplex that contains the symmetric operator sequence and incorporates 5-bromo-deoxyuridine nucleosides has been determined by multiple-wavelength anomalous diffraction phasing at 1.96-A resolution. KorA is present as a symmetric dimer and contacts DNA via a helix-turn-helix motif. Each half-site of the symmetric operator DNA binds one copy of the protein in the major groove. As confirmed by mutagenesis, recognition specificity is based on two KorA side chains forming hydrogen bonds to four bases within each operator half-site. KorA has a unique dimerization module shared by the RP4 proteins TrbA and KlcB. We propose that these proteins cooperate with the global RP4 repressor KorB in a similar manner via this dimerization module and thus regulate RP4 inheritance.

SUBMITTER: Konig B

PROVIDER: S-EPMC2665229 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Crystal structure of KorA bound to operator DNA: insight into repressor cooperation in RP4 gene regulation.

König Bettina B Müller Jürgen J JJ Lanka Erich E Heinemann Udo U

Nucleic acids research 20090203 6

KorA is a global repressor in RP4 which regulates cooperatively the expression of plasmid genes whose products are involved in replication, conjugative transfer and stable inheritance. The structure of KorA bound to an 18-bp DNA duplex that contains the symmetric operator sequence and incorporates 5-bromo-deoxyuridine nucleosides has been determined by multiple-wavelength anomalous diffraction phasing at 1.96-A resolution. KorA is present as a symmetric dimer and contacts DNA via a helix-turn-he ...[more]

PMID: 19190096

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Project description:The first-order rate constants for the RecA-independent, spontaneous, pH-dependent autocleavage of the lambda cI repressor was measured in the present study at pH 10.6 at 27, 37 and 42 degrees C respectively. Autocleavage of the repressor occurs also at pH 9 and 8, although at progressively slower rates. We demonstrate that the spontaneous autocleavage occurs also in the operator-bound state, at a rate either higher than or equal to the rate in solution, depending on the pH value. Owing to the near equality of the rate constant in both operator-free and operator-bound repressors, it can be inferred that the cleavage site has a similar structure and dynamics with respect to the catalytic site in both forms at neutral pH. Covalent modification using PMSF, brought about by a large molar excess of the reagent, inhibits autocleavage of the lambda repressor. The difficulty in obtaining this covalent modification is rationalized using our recent lambda repressor models. Bimolecular type II trans -cleavage was observed previously for mutant LexA repressors lacking a crucial catalytic serine or lysine residue [Kim and Little (1993) Cell (Cambridge, Mass.) 73, 1165-1173], but it could still be cleaved by an 85-202 'enzyme' fragment possessing an improved or hypercleavable character lacking its own cleavage site. Such a type II trans -cleavage was not observed with the covalently modified intact lambda repressor used as substrate and the purified wild-type lambda repressor 112-236 fragment used as the 'enzyme'. All these results show that for the wild-type lambda repressor, the catalytic site is close to the cleavage site in both operator-free and -bound states. In the lytic pathway, the repressor is mainly cleaved via RecA-mediated cleavage, which occurs much faster than the spontaneous autocleavage; the possible biological significance of this slow, spontaneous, but constant, autocleavage is related to the lysogenic state, when RecA-mediated cleavage is absent.

Project description:The bacterium Listeria monocytogenes ubiquitously occurs in the environment but can cause severe invasive disease in susceptible individuals when ingested. We recently identified the L. monocytogenes genes lieAB and lftRS, encoding a multidrug resistance ABC transporter and a regulatory module, respectively. These genes jointly mediate resistance against aurantimycin, an antibiotic produced by the soil-dwelling species Streptomyces aurantiacus, and thus contribute to the survival of L. monocytogenes in its natural habitat, the soil. Repression of lieAB and lftRS is exceptionally tight but strongly induced in the presence of aurantimycin. Repression depends on LftR, which belongs to subfamily 2 of the PadR-like transcriptional repressors. To better understand this interesting class of transcriptional repressors, we here deduce the LftR operator sequence from a systematic truncation and mutation analysis of the P lieAB promoter. The sequence identified is also present in the P lftRS promoter but not found elsewhere in the chromosome. Mutational analysis of the putative operator in the P lftRS promoter confirmed its relevance for LftR-dependent repression. The proposed operator sequence was sufficient for DNA binding by LftR in vitro, and a mutation in this sequence affected aurantimycin resistance. Our results provide further insights into the transcriptional adaptation of an important human pathogen to survive the conditions in its natural reservoir.IMPORTANCEListeria monocytogenes is an environmental bacterium that lives in the soil but can infect humans upon ingestion, and this can lead to severe invasive disease. Adaptation to these entirely different habitats involves massive reprogramming of transcription. Among the differentially expressed genes is the lieAB operon, which encodes a transporter for the detoxification of aurantimycin, an antimicrobial compound produced by soil-dwelling competitors. While lieAB is important for survival in the environment, its expression is detrimental during infection. We here identify critical elements in the lieAB promoter and its transcriptional regulator LftR that contribute to habitat-specific expression of the lieAB genes. These results further clarify the molecular mechanisms underlying the aurantimycin resistance of L. monocytogenes.

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Crystal structure of KorA bound to operator DNA: insight into repressor cooperation in RP4 gene regulation.

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Crystal structure of KorA bound to operator DNA: insight into repressor cooperation in RP4 gene regulation.

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