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

Abstract

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...

High-throughput and multiplexed protein array technology: protein–DNA and protein–protein interactions

“…In ArgR proteins, a winged helix-turn-helix motif (wHTH) located in the N-terminal DNA-binding domain [165,166] recognizes two adjacent 18 bp imperfect inverted repeats (Arg boxes) in operators [167,168], whereas distinct amino acids located in the C-terminal domain are responsible for the binding of l-arginine co-repressor and protein oligomerization [169]. Domain-and linker-replaced chimeric proteins were constructed from E. coli and Bacillus stearothermophilus repressors and eight proteins were compared in parallel assays (64 spots of two-fold diluted samples on each membrane) to bind operator DNAs in the presence and the absence of arginine [163]. The detected signal intensity correlated with the DNA-binding affinity as confirmed by EMSA and SPR.…”

“…Next, this highly sensitive method was applied to a comprehensive analysis of the ArgR-mediated regulatory system in distant mesophilic and thermophilic bacteria [163,164]. In ArgR proteins, a winged helix-turn-helix motif (wHTH) located in the N-terminal DNA-binding domain [165,166] recognizes two adjacent 18 bp imperfect inverted repeats (Arg boxes) in operators [167,168], whereas distinct amino acids located in the C-terminal domain are responsible for the binding of l-arginine co-repressor and protein oligomerization [169].…”

Similarity and divergence between the RNA polymerase α subunits from hyperthermophilic Thermotoga maritima and mesophilic Escherichia coli bacteria

“…We used the protein array method by applying near-infrared fluorescent DNA probes for the detection of molecular interactions as described previously (Ghochikyan et al, 2002;Snapyan et al, 2003). Serial dilutions of the purified His-tagged α Tm of T. maritima and ArgR proteins of T. neapolitana and B. stearothermophilus (used as positive controls) and E. coli (used as a negative control) were spotted on a nitrocellulose membrane and incubated with two IRDye-labeled DNA probes, a full-length PargGo promoter-operator DNA or its shorter derivative PargGoDA/T DNA that lacks a potential UP-element (Fig.…”

Competition on Nitrocellulose-immobilized Antibody Arrays

“…To evaluate the onecolor and two-color detection methods in protein profiling with antibodies arrayed on a nitrocellulose (NC) membrane, we have chosen near-infrared fluorescent dyes (IRDyes) for labeling proteins. Near-infrared fluorescence provides high sensitivity on membrane supports (17) and has been successfully used to detect molecular interactions on arrayed purified proteins (18), phage-displayed peptides (19), rat neuronal membrane fractions (20), and crude prokaryotic extracts (21). As the Escherichia coli ␣ subunit of RNAP (␣RNAP), labeled by IRDye, retains its binding ability to arrayed transcriptional factors (21), it has been used as a reporter to assess the competitiveness of labeled and unlabeled molecules in binding assays.…”