Severe MgADP inhibition of Bacillus subtilis F1-ATPase is not due to the absence of nucleotide binding to the noncatalytic nucleotide binding sites.
Ontology highlight
ABSTRACT:
SUBMITTER: Ishikawa T
PROVIDER: S-EPMC4171097 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ? subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ? subunit. The ? subunit did not inhibit but activated BF1. We conclude that the ? subunit relieves BF1 from MgADP inhibition.
| S-EPMC3742539 | biostudies-literature
Project description:The CodY protein is a global transcriptional regulator that controls, directly or indirectly, expression of more than 100 genes and operons in Bacillus subtilis. We used in vitro DNA affinity purification combined with massively parallel sequencing, to identify B. subtilis chromosomal DNA fragments that bind CodY in vitro. A nonstandard strand-specific analysis of the data allowed us to pinpoint CodY-binding sites at single-nucleotide resolution. By comparing the extent of binding at decreasing CodY concentrations, we were able to classify binding regions according to their relative strengths and construct a subset of the 323 strongest CodY-binding regions that included sites associated with nearly all genes reported to be direct CodY targets. Many of the identified sites were located within coding regions. At such sites within the ispA, rapA, and rapE genes CodY-dependent repression was demonstrated using lacZ fusions and mutational analysis.
| S-EPMC3637721 | biostudies-literature
Project description:The transcriptional factor Zur plays a key role in regulating zinc homeostasis in Bacillus subtilis. The genomic sites bound by Zur were mapped using a chromatine immunoprecipitation approach. This allowed the identification of 80 inter- and intragenic chromosomal sites bound by Zur.
2015-01-06 | GSE64671 | GEO
Project description:CodY is a global transcriptional regulator that is known to control directly the expression of at least two dozen operons in Bacillus subtilis, but the rules that govern the binding of CodY to its target DNA have been unclear. Using DNase I footprinting experiments, we identified CodY-binding sites upstream of the B. subtilis ylmA and yurP genes. The protected regions overlapped versions of a previously proposed CodY-binding consensus motif, AATTTTCWGAAAATT. Multiple single mutations were introduced into the CodY-binding sites of the ylmA, yurP, dppA, and ilvB genes. The mutations affected both the affinity of CodY for its binding sites in vitro and the expression in vivo of lacZ fusions that carry these mutations in their promoter regions. Our results show that versions of the AATTTTCWGAAAATT motif, first identified for Lactococcus lactis CodY, with up to five mismatches play an important role in the interaction of B. subtilis CodY with DNA.
| S-EPMC2238193 | biostudies-literature
Project description:High-resolution crystallographic studies of a number of inhibited forms of bovine F1-ATPase have identified four independent types of inhibitory site: the catalytic site, the aurovertin B-binding site, the efrapeptin-binding site and the site to which the natural inhibitor protein IF1 binds. Hitherto, the binding sites for other inhibitors, such as polyphenolic phytochemicals, non-peptidyl lipophilic cations and amphiphilic peptides, have remained undefined. By employing multiple inhibition analysis, we have identified the binding sites for these compounds. Several of them bind to the known inhibitory sites. The amphiphilic peptides melittin and synthetic analogues of the mitochondrial import pre-sequence of yeast cytochrome oxidase subunit IV appear to mimic the natural inhibitor protein, and the polyphenolic phytochemical inhibitors resveratrol and piceatannol compete for the aurovertin B-binding site (or sites). The non-peptidyl lipophilic cation rhodamine 6G acts at a separate unidentified site, indicating that there are at least five inhibitory sites in the F1-ATPase. Each of the above inhibitors has significantly different activity against the bacterial Bacillus PS3 alpha3beta3gamma subcomplex compared with that observed with bovine F1-ATPase. IF1 does not inhibit the bacterial enzyme, even in the absence of the epsilon-subunit. An understanding of these inhibitors may enable rational development of therapeutic agents to act as novel antibiotics against bacterial ATP synthases or for the treatment of several disorders linked to the regulation of the ATP synthase, including ischaemia-reperfusion injury and some cancers.
| S-EPMC1134879 | biostudies-literature
Project description:The transcriptional factor Zur plays a key role in regulating zinc homeostasis in Bacillus subtilis. The genomic sites bound by Zur were mapped using a chromatine immunoprecipitation approach. This allowed the identification of 80 inter- and intragenic chromosomal sites bound by Zur. This data set contains 2 samples. Immunoprecipitated (IP) DNA from Bacillus subtilis BSAS36 strain (BSB1, a tryptophan-prototrophic derivative 168 strain expressing a SPA-tagged Zur protein) were extracted from bacterial cells in the exponential growth phase. IP and whole-cell extract DNA were hybridized on tiling array to generate ChIP-on-chip profiles. Two biological replicates were anlyzed.
2015-01-06 | E-GEOD-64671 | biostudies-arrayexpress
Project description:Molecular machines fueled by NTP play pivotal roles in a wide range of cellular activities. One common feature among NTP-driven molecular machines is that NTP binding is a major force-generating step among the elementary reaction steps comprising NTP hydrolysis. To understand the mechanism in detail,in this study, we conducted a single-molecule rotation assay of the ATP-driven rotary motor protein F1-ATPase using uridine triphosphate (UTP) and a base-free nucleotide (ribose triphosphate) to investigate the impact of a pyrimidine base or base depletion on kinetics and force generation. Although the binding rates of UTP and ribose triphosphate were 10(3) and 10(6) times, respectively, slower than that of ATP, they supported rotation, generating torque comparable to that generated by ATP. Affinity change of F1 to UTP coupled with rotation was determined, and the results again were comparable to those for ATP, suggesting that F1 exerts torque upon the affinity change to UTP via rotation similar to ATP-driven rotation. Thus, the adenine-ring significantly enhances the binding rate, although it is not directly involved in force generation. Taking into account the findings from another study on F1 with mutated phosphate-binding residues, it was proposed that progressive bond formation between the phosphate region and catalytic residues is responsible for the rotation-coupled change in affinity.
| S-EPMC4119285 | biostudies-literature
Project description:The crystal structure of nucleotide-free yeast F(1) ATPase has been determined at a resolution of 3.6 A. The overall structure is very similar to that of the ground state enzyme. In particular, the beta(DP) and beta(TP) subunits both adopt the closed conformation found in the ground state structure despite the absence of bound nucleotides. This implies that interactions between the gamma and beta subunits are as important as nucleotide occupancy in determining the conformational state of the beta subunits. Furthermore, this result suggests that for the mitochondrial enzyme, there is no state of nucleotide occupancy that would result in more than one of the beta subunits adopting the open conformation. The adenine-binding pocket of the beta(TP) subunit is disrupted in the apoenzyme, suggesting that the beta(DP) subunit is responsible for unisite catalytic activity.
| S-EPMC2667741 | biostudies-literature
Project description:In Bacillus subtilis, two major transcriptional factors, GlnR and TnrA, are involved in a sophisticated network of adaptive responses to nitrogen availability. GlnR was reported to repress the transcription of the glnRA, tnrA and ureABC operons under conditions of excess nitrogen. As GlnR and TnrA regulators share the same DNA binding motifs, a genome-wide mapping of in vivo GlnR-binding sites was still needed to clearly define the set of GlnR/TnrA motifs directly bound by GlnR.We used chromatin immunoprecipitation coupled with hybridization to DNA tiling arrays (ChIP-on-chip) to identify the GlnR DNA-binding sites, in vivo, at the genome scale.We provide evidence that GlnR binds reproducibly to 61 regions on the chromosome. Among those, 20 regions overlap the previously defined in vivo TnrA-binding sites. In combination with real-time in vivo transcriptional profiling using firefly luciferase, we identified the alsT gene as a new member of the GlnR regulon. Additionally, we characterized the GlnR secondary regulon, which is composed of promoter regions harboring a GlnR/TnrA box and bound by GlnR in vivo. However, the growth conditions revealing a GlnR-dependent regulation for this second category of genes are still unknown.Our findings show an extended overlap between the GlnR and TnrA in vivo binding sites. This could allow efficient and fine tuning of gene expression in response to nitrogen availability. GlnR appears to be part of complex transcriptional regulatory networks, which involves interactions between different regulatory proteins.
| S-EPMC5569456 | biostudies-literature
Project description:The enzyme F1-adenosine triphosphatase (ATPase) is a molecular motor that converts the chemical energy stored in the molecule adenosine triphosphate (ATP) into mechanical rotation of its gamma-subunit. During steady-state catalysis, the three catalytic sites of F1 operate in a cooperative fashion such that at every instant each site is in a different conformation corresponding to a different stage along the catalytic cycle. Notwithstanding a large amount of biochemical and, recently, structural data, we still lack an understanding of how ATP hydrolysis in F1 is coupled to mechanical motion and how the catalytic sites achieve cooperativity during rotatory catalysis. In this publication, we report combined quantum mechanical/molecular mechanical simulations of ATP hydrolysis in the betaTP and betaDP catalytic sites of F1-ATPase. Our simulations reveal a dramatic change in the reaction energetics from strongly endothermic in betaTP to approximately equienergetic in betaDP. The simulations identify the responsible protein residues, the arginine finger alphaR373 being the most important one. Similar to our earlier study of betaTP, we find a multicenter proton relay mechanism to be the energetically most favorable hydrolysis pathway. The results elucidate how cooperativity between catalytic sites might be achieved by this remarkable molecular motor.
| S-EPMC1304769 | biostudies-literature