DNA binding of <i>Escherichia coli</i> arginine repressor mutants altered in oligomeric state

Chen, Sheau-Hu; Merican, Amir Feisal; Sherratt, David J.

doi:10.1046/j.1365-2958.1997.4301791.x

Cited by 30 publications

(38 citation statements)

References 21 publications

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“…DNA-protein interactions are highly dynamic processes that depend on both partners, and local folding transition of a transcription-regulatory protein can be coupled to site-specific DNA binding (37). DNA-binding affinity varies for different arg-specific operators and is considerably reduced for a single Arg box of E. coli operators (6,8,42,43). A single Arg box has been identified for several arginine-related and nonrelated genes (13,20,25,26,30,38), though binding of ArgR to these targets has not yet been proven.…”

Section: Discussionmentioning

confidence: 99%

“…Histagged wild-type and chimeric ArgR proteins were purified with minor modifications as follows. Bacterial cells were suspended in a buffer (50 mM NaH 2 PO 4 [pH 8.0], 300 mM NaCl, 10 mM imidazole) and sonicated, and cell extracts were subjected to affinity chromatography on a nickel-nitrilotriacetic acid column (Qiagen). The column was equilibrated and washed with the buffer defined above, and ArgR proteins were eluted with the same buffer containing 250 mM imidazole.…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, arginine binding to distinct amino acids (four of the six residues involved are conserved in the oligomerization domain of both ArgR proteins; Fig. 1) leads to allosteric changes in both hexameric repressors (28,41,45), thereby increasing their arg operator DNA-binding affinity (5,6,8,10,21,42,43,46). It has been shown that binding six arginine molecules, in addition to reinforcing interactions between monomers within a trimer FIG.…”

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confidence: 98%

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Arginine Operator Binding by Heterologous and Chimeric ArgR Repressors fromEscherichia coliandBacillus stearothermophilus

et al. 2002

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Bacillus stearothermophilus ArgR binds efficiently to the Escherichia coli carAB operator, whereas the E. coli repressor binds very poorly to the argCo operator of B. stearothermophilus. In order to elucidate this contradictory behavior between ArgRs, we constructed chimeric proteins by swapping N-terminal DNA-binding and C-terminal oligomerization domains or by exchanging the linker peptide. Chimeras carrying the E. coli DNA-binding domain and the B. stearothermophilus oligomerization domain showed sequence-nonspecific rather than sequence-specific interactions with arg operators. Chimeras carrying the B. stearothermophilus DNA-binding domain and E. coli oligomerization domain exhibited a high DNA-binding affinity for the B. stearothermophilus argCo and E. coli carAB operators and repressed the reporter-gene transcription from the B. stearothermophilus PargCo control region in vitro; arginine had no effect on, and indeed even decreased, their DNA-binding affinity. With the protein array method, we showed that the wild-type B. stearothermophilus ArgR and derivatives of it containing only the exchanged linker from E. coli ArgR or carrying the B. stearothermophilus DNA-binding domain along with the linker and the ␣4 regions were able to bind argCo containing the single Arg box. This binding was weaker than binding to the two-box operator but was no longer arginine dependent. Several lines of observations indicate that the ␣4 helix in the oligomerization domain and the linker peptide can contribute to the recognition of single or double Arg boxes and therefore to the operator DNA-binding specificity in similar but not identical ArgR repressors from two distant bacteria.Recent genome comparative analysis has revealed a global vision of arg genes transcription regulation in microorganisms (3,23,26,30). However, experimental studies on distant organisms are required to elucidate the molecular mechanisms and to understand the evolutionary principles established for arginine regulatory systems in various microbes.A substantial amount of functional and structural information regarding transcription regulation has accumulated for the Escherichia coli arginine repressor, ArgR (14,22,40). In this organism, the repressor, in cooperation with L-arginine, governs expression of the arginine biosynthesis genes (arg) and carAB genes, coding for carbamoylphosphate synthetase (EC 6.3.5.5), providing the carbamoylphosphate required for the synthesis of both arginine and pyrimidine residues. The ArgR protein consists of N-terminal DNA-binding and C-terminal oligomerization domains connected by a short protease-sensitive linker (15), and a three-dimensional structure has been separately resolved for each domain (41,45). A winged helixturn-helix (wHTH) motif of the DNA-binding domain (41) recognizes a 40-bp sequence which comprises two adjacent imperfect 18-bp palindromes, known as Arg boxes, that are separated by a 3-bp spacer in the majority of cognate operators or by a 2-bp spacer in the argR operator (6,44,46). ArgR monomers associ...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 98%

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Arginine Operator Binding by Heterologous and Chimeric ArgR Repressors fromEscherichia coliandBacillus stearothermophilus

et al. 2002

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“…ArgR is a hexamer in solution and normally binds as a hexamer to two closely spaced Arg-boxes present in the promoter regions of arginine biosynthetic genes (Glansdorff, 1996). However, ArgR binds as a hexamer to a single Arg-box within cer and imposes a bend of approximately 65Њ Chen et al, 1997). It has been proposed that, as well as imposing a structurally important bend in cer accessory DNA, ArgR forms a bridge between two recombining cer sites (Alé n et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

The ArcA/ArcB two‐component regulatory system of Escherichia coli is essential for Xer site‐specific recombination at psi

Colloms

Alén

Sherratt

1998

Molecular Microbiology

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SummaryTwo recombinases, XerC and XerD, act at the recombination sites psi and cer in plasmids pSC101 and ColE1 respectively. Recombination at these sites maintains the plasmids in a monomeric state and helps to promote stable plasmid inheritance. The accessory protein PepA acts at both psi and cer to ensure that only intramolecular recombination takes place. An additional accessory protein, ArgR, is required for recombination at cer but not at psi. Here, we demonstrate that the ArcA /ArcB two-component regulatory system of Escherichia coli, which mediates adaptation to anaerobic growth conditions, is required for efficient recombination in vivo at psi. Phosphorylated ArcA binds to psi in vitro and increases the efficiency of recombination at this site. Binding of ArcA to psi may contribute to the formation of a higher order synaptic complex between a pair of psi sites, thus helping to ensure that recombination is intramolecular.

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“…The arginine-promoted dimerization of trimers appears to be a crucial step for the formation of a functional repressor, since the arginine-bound form of the E. coli ArgR hexamer displays a higher anity for operator DNAs than the free hexamer or trimer (Burke et al 1994;Tian et al 1994). In the presence of arginine the hexameric ArgR binds to DNA operators containing four Arg box half-sites with higher anity than to those containing two or three half-sites (Chen et al 1997). The arginine-bound form of the hexameric E. coli ArgR covers four turns of the operator DNA and makes tight contacts with four segments of the major and two segments of the minor grooves within the operator sequence .…”

Section: Introductionmentioning

confidence: 99%

Thermostability, oligomerization and DNA-binding properties of the regulatory protein ArgR from the hyperthermophilic bacterium Thermotoga neapolitana

et al. 2000

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The hexameric regulatory protein ArgR formed by arginine-mediated dimerization of identical trimers governs the expression of genes required for arginine metabolism and some other genes in mesophilic and moderately thermophilic bacteria. We have cloned the argR gene from two hyperthermophilic bacteria of the genus Thermotoga. The two-domain ArgR proteins encoded by T. neapolitana and T. maritima share a low degree of sequence similarity with other bacterial arginine repressors. The ArgR protein from T. neapolitana binds to an operator located just upstream of its coding sequence and, therefore, the argR gene may be autoregulated. The protein has extremely high intrinsic thermostability and tolerance to urea. Moreover, its binding to target DNA increases the melting temperature by approximately 15°C. The formation of oligomeric ArgR-DNA complexes is a function of protein concentration, with hexameric complexes being favoured at higher concentrations. In the presence of arginine the hyperthermophilic ArgR protein binds to its own operator, argRo, only by forming hexamer ArgR-DNA complexes, whereas both trimer-DNA and hexamer-DNA complexes are detected in the absence of arginine. However, the anity of T. neapolitana ArgR for DNA has been found to be higher for a mixture of trimers and non-bound hexamers than for argininebound hexamers. Our data indicate that genes for arginine biosynthesis are clustered in a putative operon, which could also be regulated by the ArgR protein, in the hyperthermophilic host.

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DNA binding of Escherichia coli arginine repressor mutants altered in oligomeric state

Cited by 30 publications

References 21 publications

Arginine Operator Binding by Heterologous and Chimeric ArgR Repressors fromEscherichia coliandBacillus stearothermophilus

Arginine Operator Binding by Heterologous and Chimeric ArgR Repressors fromEscherichia coliandBacillus stearothermophilus

The ArcA/ArcB two‐component regulatory system of Escherichia coli is essential for Xer site‐specific recombination at psi

Thermostability, oligomerization and DNA-binding properties of the regulatory protein ArgR from the hyperthermophilic bacterium Thermotoga neapolitana

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