Protein component of Bacillus subtilis RNase P specifically enhances the affinity for precursor-tRNAAsp

Ribonuclease P (RNase P) is an endonuclease that cleaves precursor tRNA to form the 5'-end of mature tRNA and is composed of a catalytic RNA subunit and a small protein subunit. The function of the protein component of Bacillus subtilis RNase P in catalysis of B. subtilis precursor tRNAAsp cleavage has been elucidated using steady-state kinetics, transient kinetics, and ligand affinity measurements to compare the functional properties of RNase P holoenzyme to RNase P RNA in 10 mM MgCl2, 100 mM NH4Cl. The protein component modestly affects several steps including 相似文献   

Phosphorylation on histidine is accompanied by localized structural changes in the phosphocarrier protein, HPr from Bacillus subtilis

The histidine-containing protein (HPr) of bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) serves a central role in a series of phosphotransfer reactions used for the translocation of sugars across cell membranes. These studies report the high-definition solution structures of both the unphosphorylated and histidine phosphorylated (P-His) forms of HPr from Bacillus subtilis. Consistent with previous NMR studies, local conformational adjustments occur upon phosphorylation of His 15, which positions the phosphate group to serve as a hydrogen bond acceptor for the amide protons of Ala 16 and Arg 17 and to interact favorably with the alpha-helix macrodipole. However, the positively charged side chain of the highly conserved Arg 17 does not appear to interact directly with phospho-His 15, suggesting that Arg 17 plays a role in the recognition of other PTS enzymes or in phosphotransfer reactions directly. Unlike the results reported for Escherichia coli P-His HPr (Van Nuland NA, Boelens R, Scheek RM, Robillard GT, 1995, J Mol Biol 246:180-193), our data indicate that phosphorylation of His 15 is not accompanied by adoption of unfavorable backbone conformations for active site residues in B. subtilis P-Ser HPr.     

Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci

The rnc gene of Bacillus subtilis, which has 36% amino acid identity with the gene that encodes Escherichia coli RNase III endonuclease, was cloned in E. coli and shown by functional assays to encode B. subtilis RNase III (Bs-RNase III). The cloned B. subtilis rnc gene could complement an E. coli rnc strain that is deficient in rRNA processing, suggesting that Bs-RNase III is involved in rRNA processing in B. subtilis. Attempts to construct a B. subtilis rnc null mutant were unsuccessful, but a strain was constructed in which only a carboxy-terminal truncated version of Bs-RNase III was expressed. The truncated Bs-RNase III showed virtually no activity in vitro but was active in vivo. Analysis of expression of a copy of the rnc gene integrated at the amy locus and transcribed from a p(spac) promoter suggested that expression of the B. subtilis rnc is under regulatory control.     

Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis

Ribonuclease P is a ribonucleoprotein complex that catalyzes the essential 5' maturation of all precursor tRNA molecules. The protein component both alters the conformation of the RNA component and enhances the substrate affinity and specificity. To facilitate biochemical and biophysical studies, the protein component of Bacillus subtilis ribonuclease P (RNase P) was overproduced in Escherichia coli using the native amino acid sequence with the initial 20 codons optimized for expression in E.coli . A simple purification procedure using consecutive cation exchange chromatography steps in the presence and absence of urea was developed to purify large quantities of P protein without contaminating nucleic acids. The identity of the recombinant protein as a cofactor of RNase P was established by its ability to stimulate the activity of the RNA component in low ionic strength buffer in a 1:1 stoichiometry. Circular dichroism studies indicate that P protein is a combination of alpha-helix and beta-sheet secondary structures and is quite stable, with a T m of 67 degrees C. The described methods facilitated the large scale purification of homogeneous, RNA-free P protein required for high resolution crystallographic analyses and may be useful for the preparation of other RNA binding proteins.     

Interaction of substrate and effector binding sites in the ArsA ATPase

The ars operon of plasmid R773 confers resistance to antimonials and arsenicals in Escherichia coli by encoding an ATP-dependent extrusion system for the oxyanions. The catalytic subunit, the ArsA protein, is an ATPase with two nucleotide binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein. The ArsA ATPase is allosterically activated by tricoordinate binding of As(3+) or Sb(3+) to three cysteine thiolates. Previous measurements suggested that the intrinsic fluorescence of tryptophans might be useful for examining binding of Mg2+ ATP and antimonite. In the present study an increase in intrinsic tryptophan fluorescence was observed upon addition of Mg2+ ATP. This enhancement was reversed by addition of antimonite. The ArsA protein contains four tryptophan residues: Trp159, Trp253, Trp522, and Trp524. The first two were altered to tyrosine residues by site-directed mutagenesis. Cells expressing both the arsAW159Y and arsAW253Y mutations retained resistance to arsenite, and the purified W159Y and W253Y proteins retained ATPase activity. While the intrinsic tryptophan fluorescence of the W253Y protein responded to addition of Mg2+ ATP, intrinsic tryptophan fluorescence in the purified W159Y protein was no longer enhanced by substrate. These results suggest that Trp159 is conformationally coupled to one or both of the nucleotide binding sites and provides a useful probe for the interaction of effector and substrate binding sites.     

Peptidoglycan structural dynamics during germination of Bacillus subtilis 168 endospores

Peptidoglycan structural dynamics during endospore germination of Bacillus subtilis 168 have been examined by muropeptide analysis. The first germination-associated peptidoglycan structural changes are detected within 3 min after the addition of the specific germinant L-alanine. We detected in the spore-associated material new muropeptides which, although they have slightly longer retention times by reversed-phase (RP)-high-pressure liquid chromatography (HPLC) than related ones in dormant spores, show the same amino acid composition and molecular mass. Two-dimensional nuclear magnetic resonance (NMR) analysis shows that the chemical changes to the muropeptides on germination are minor and are probably limited to stereochemical inversion. These new muropeptides account for almost 26% of the total muropeptides in spore-associated material after 2 h of germination. The exudate of germinated spores of B. subtilis 168 contains novel muropeptides in addition to those present in spore-associated material. Exudate-specific muropeptides have longer retention times, have no reducing termini, and exhibit a molecular mass 20 Da lower than those of related reduced muropeptides. These new products are anhydro-muropeptides which are generated by a lytic transglycosylase, the first to be identified in a gram-positive bacterium. There is also evidence for the activity of a glucosaminidase during the germination process. Quantification of muropeptides in spore-associated material indicates that there is a heterogeneous distribution of muropeptides in spore peptidoglycan. The spore-specific residue, muramic delta-lactam, is proposed to be a major substrate specificity determinant of germination-specific lytic enzymes, allowing cortex hydrolysis without any effect on the primordial cell wall.     

Improved binding of cytochrome P450cam substrate analogues designed to fill extra space in the substrate binding pocket

Cytochrome P450cam catalyzes the 5-exo-hydroxylation of camphor. Camphor analogues were designed to fill an empty region of the substrate binding pocket with the expectation that they would bind more tightly than camphor itself due to increased van der Waals interactions with the protein and the displacement of any solvent occupying this site. A series of compounds (endo-borneol methyl ether, endo-borneol propyl ether, endo-borneol allyl ether and endo-borneol dimethyl allyl ether) were synthesized with substituents at the camphor carbonyl oxygen. The spin conversion and thermodynamic properties of this series of compounds were measured for wild type and Y96F mutant cytochrome P450cam and were interpreted in the context of molecular dynamics simulations of the camphor analogues in the P450 binding site and in solution. Compounds with a 3-carbon chain substituent were predicted to match the size of the unoccupied region most optimally and thus bind best. Consistent with this prediction, the borneol allyl ether binds to cytochrome P450cam with highest affinity with a Kd = 0.6 +/- 0.1 microM (compared to a Kd = 1.7 +/- 0.2 microM for camphor under the same experimental conditions). Binding of the camphor analogues to the Y96F mutant is much enhanced over the binding of camphor, indicating that hydrogen bonding plays a less important role in binding of these analogues. Binding enthalpies calculated from the simulations, taking all solvent contributions into account, agree very well with experimental binding enthalpies. Binding affinity is not however correlated with the calculated binding enthalpy because the binding of the substrate analogues is characterized by enthalpy-entropy compensation. The new compounds are useful probes for further studies of the mechanism of cytochrome P450cam due to their high binding affinities and high spin properties.     

Identification of the genes encoding Mn2+-dependent RNase HII and Mg2+-dependent RNase HIII from Bacillus subtilis: classification of RNases H into three families

Database searches indicated that the genome of Bacillus subtilis contains three different genes encoding RNase H homologues. The ypdQ gene encodes an RNase HI homologue with 132 amino acid residues, whereas the rnh and ysgB genes encode RNase HII homologues with 255 and 313 amino acid residues, respectively. RNases HI and HII show no significant sequence similarity. These genes were individually expressed in Escherichia coli; the recombinant proteins were purified, and their enzymatic properties were compared with those of E. coli RNases HI and HII. We found that the ypdQ gene product showed no RNase H activity. The 2.2 kb pair genomic DNA containing this gene did not suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that this gene product shows no RNase H activity in vivo as well. In contrast, the rnh (rnhB) gene product (RNase HII) showed a preference for Mn2+, as did E. coli RNase HII, whereas the ysgB (rnhC) gene product (RNase HIII) exhibited a Mg2+-dependent RNase H activity. Oligomeric substrates digested with these enzymes indicate similar recognition of these substrates by B. subtilis and E. coli RNases HII. Likewise, B. subtilis RNase HIII and E. coli RNase HI have generated similar products. These results suggest that B. subtilis RNases HII and HIII may be functionally similar to E. coli RNases HII and HI, respectively. We propose that Mn2+-dependent RNase HII is universally present in various organisms and Mg2+-dependent RNase HIII, which may have evolved from RNase HII, functions as a substitute for RNase HI.     

Spectral and cyanide binding properties of the cytochrome aa3 (600 nm) complex from Bacillus subtilis

The cytochrome aa3 (600 nm) complex, or menaquinol oxidase, from Bacillus subtilis is a member of the cytochrome oxidase superfamily of respiratory membrane protein complexes. We have characterized some spectral properties of this enzyme and its reaction with cyanide. The magnetic circular dichroism (MCD) spectrum of the oxidized enzyme has a single band at 1560 nm in the near-infrared region assigned to bis-histidine-ligated, low-spin ferricytochrome a. The other heme, cytochrome a3, is presumably high-spin in the oxidized enzyme, as isolated. The absence of a trough in the MCD spectrum at 790 nm, observed previously with mammalian cytochrome c oxidase and assigned to CuA (Greenwood et al., Biochem. J. 215, 303-316, 1983), is consistent with the absence of this center from the menaquinol oxidase. When the heme ligand cyanide is added to oxidized menaquinol oxidase, a new MCD band appears at 2010 nm, while the band at 1560 nm is unperturbed. The new band is assigned to low-spin ferricytochrome a3 bound with cyanide. The long-wavelength position of this cyanide-induced band is proposed to arise from the close interaction of cytochrome a3 with the copper atom, CuB. The kinetics of cyanide binding to oxidized cytochrome aa3(600 nm) reveal a spectrally simple, yet kinetically complex process. The reaction is biphasic with second-order rate constants of 45 and 0.61 M-1s-1 at 1 mM KCN, with each phase constituting about 50% of the overall reaction. When the enzyme is subjected to a cycle of anaerobic reduction and air oxidation, the subsequent reaction with cyanide occurs in a single phase at the faster rate. This behavior is ascribed to different conformations of the binuclear center exhibiting different reactivities with cyanide, and is in keeping with that previously established for the structurally more complex mitochondrial cytochrome c oxidase. However, the electronic spectral characteristics of some of the species involved in these reactions are different in the present bacterial case from those of reported eukaryotic systems.     

Inhibition of the hammerhead ribozyme cleavage reaction by site-specific binding of Tb

Terbium(III) [Tb(III)] was shown to inhibit the hammerhead ribozyme by competing with a single magnesium(II) ion. X-ray crystallography revealed that the Tb(III) ion binds to a site adjacent to an essential guanosine in the catalytic core of the ribozyme, approximately 10 angstroms from the cleavage site. Synthetic modifications near this binding site yielded an RNA substrate that was resistant to Tb(III) binding and capable of being cleaved, even in the presence of up to 20 micromolar Tb(III). It is suggested that the magnesium(II) ion thought to bind at this site may act as a switch, affecting the conformational changes required to achieve the transition state.     

Substitution of the C-terminal domain of the Escherichia coli RNA polymerase alpha subunit by that from Bacillus subtilis makes the enzyme responsive to a Bacillus subtilis transcriptional activator

Cyanobacteria have two protochlorophyllide (Pchlide) reductases catalyzing the conversion of Pchlide to chlorophyllide, a key step in the biosynthetic pathway of chlorophylls (Chls); a light-dependent (LPOR) and a light-independent (DPOR) reductase. We found an open reading frame (ORF322) in a 2,131-bp EcoRI fragment from the genomic DNA of the cyanobacterium Plectonema boryanum. Because the deduced amino acid sequence showed a high similarity to those of various plant LPORs and the LPOR activity was detected in the soluble fraction of Escherichia coli cells over-expressing the ORF322 protein, ORF322 was defined as the por gene encoding LPOR in P. boryanum. A por-disrupted mutant, YFP12, was isolated by targeted mutagenesis to investigate the physiological importance of LPOR. YFP12 grew as well as wild type under low light conditions (10-25 muE m-2 s-1). However, its growth was significantly retarded as a result of a significant decrease in its Chl content under higher light conditions (85-130 muE m-2 s-1). Furthermore, YFP12 stopped growing and suffered from photobleaching under the highest light intensity (170 muE m-2 s-1). In contrast, a chlL-disrupted (DPOR-less) mutant YFC2 grew as well as wild type irrespective of light intensity. From these phenotypic characteristics, we concluded that, although both LPOR and DPOR contribute to Chl synthesis in the cells growing in the light, the extent of the contribution by LPOR increases with increasing light intensity; without it, the cells are unable to grow under light intensities of more than 130 muE m-2 s-1.     

Bacillus subtilis DnaG primase stabilises the bacteriophage SPP1 G40P helicase-ssDNA complex

We analyzed our treatment results in 153 patients with histologically verified intracranial germ cell tumors and proposed classifying them into three therapeutic groups with good prognosis, intermediate prognosis, and poor prognosis. In this work, we selected patients treated with chemotherapy (cisplatin or carboplatin combinations) in each subgroup, and we discuss the role of chemotherapy in their treatment. Our combination chemotherapy regimens are: cisplatin-vinblastine-bleomycin, cisplatin-etoposide, and carboplatin-etoposide. We delivered these chemotherapies to the last 33 patients and compared their treatment results with those obtained in the previous 31 patients, who were treated with conventional radiation therapy alone. A combination with chemotherapy and a reduced dose of irradiation with local field was given to 7 patients with germinoma to increase the cure rate and reduce radiation-induced side effects, including anterior pituitary dysfunction. We obtained an excellent initial response to chemotherapy. The chemotherapy we delivered had significantly better effects in the group with intermediate prognosis, but not in the group with poor prognosis. More aggressive chemotherapy and radiation therapy should be given as the initial treatment.     

A model of the quaternary structure of enolases, based on structural and evolutionary analysis of the octameric enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pHo) for activity is 8.1-8.2, and the Km for 2-PGA is approximately 0.67 mM. Using the dimeric Calpha structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86-96) and the loop between helix L and strand 1 (HL-S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL-S1 loop is truncated by 4-6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL-S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.     

The P15-loop of Escherichia coli RNase P RNA is an autonomous divalent metal ion binding domain

We have studied the structure and divalent metal ion binding of a domain of the ribozyme RNase P RNA that is involved in base pairing with its substrate. Our data suggest that the folding of this internal loop, the P15-loop, is similar irrespective of whether it is part of the full-length ribozyme or part of a model RNA molecule. We also conclude that this element constitutes an autonomous divalent metal ion binding domain of RNase P RNA and our data suggest that certain specific chemical groups within the P15-loop participate in coordination of divalent metal ions. Substitutions of the Sp- and Rp-oxygens with sulfur at a specific position in this loop result in a 2.5-5-fold less active ribozyme, suggesting that Mg2+ binding at this position contributes to function. Our findings strengthen the concept that small RNA building blocks remain basically unchanged when removed from their structural context and thus can be used as models for studies of their potential function and structure within native RNA molecules.     

The stability of mRNA from the gsiB gene of Bacillus subtilis is dependent on the presence of a strong ribosome binding site

The term 'periventricular leukomalacia' (PVL) usually covers necrotic and/or gliotic lesions from perinatal origin occurring in the periventricular ring of telencephalic white matter. Carrying motor and neuropsychological consequences, PVLs could be the most severe danger for very premature brains. Positive rolandic sharp waves recorded on EEG and precocious abnormally echogenous periventricular images on ultrasound suggest prospective periventricular cysts. Cystic periventricular cavitations certify the diagnosis of PVL. More subtle lesions of PVL do not reach the cystic grade and their diagnosis is confirmed by MRI. Treatment of infections is already available and potentially a tool for prevention. When the overwhelming glutamatergic signal has been triggered, neuroprotective agents turning off the excitotoxic cascade, including calcium blockers, growth factors and others, are promising therapeutic tools.