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genetic and biochemical analysis of yeast and human cap trimethylguanosine synthase: functional overlap of 2,2,7-trimethylguanosine caps, small nuclear ribonucleoprotein components, pre-mrna splicing factors, and rna decay pathways.trimethylguanosine synthase (tgs1) is the enzyme that converts standard m(7)g caps to the 2,2,7-trimethylguanosine (tmg) caps characteristic of spliceosomal small nuclear rnas. fungi and mammalian somatic cells are able to grow in the absence of tgs1 and tmg caps, suggesting that an essential function of the tmg cap might be obscured by functional redundancy. a systematic screen in budding yeast identified nonessential genes that, when deleted, caused synthetic growth defects with tgs1delta. the ...200818775984
a novel mutator of escherichia coli carrying a defect in the dgt gene, encoding a dgtp triphosphohydrolase.a novel mutator locus in escherichia coli was identified from a collection of random transposon insertion mutants. several mutators in this collection were found to have an insertion in the dgt gene, encoding a previously characterized dgtp triphosphohydrolase. the mutator activity of the dgt mutants displays an unusual specificity. among the six possible base pair substitutions in a lacz reversion system, the g.c-->c.g transversion and a.t-->g.c transition are strongly enhanced (10- to 50-fold) ...200818776019
editing of misaligned 3'-termini by an intrinsic 3'-5' exonuclease activity residing in the php domain of a family x dna polymerase.bacillus subtilis gene yshc encodes a family x dna polymerase (polx(bs)), whose biochemical features suggest that it plays a role during dna repair processes. here, we show that, in addition to the polymerization activity, polx(bs) possesses an intrinsic 3'-5' exonuclease activity specialized in resecting unannealed 3'-termini in a gapped dna substrate. biochemical analysis of a polx(bs) deletion mutant lacking the c-terminal polymerase histidinol phosphatase (php) domain, present in most of the ...200818776221
characterization of a unique clpb protein of mycoplasma pneumoniae and its impact on growth.mycoplasma pneumoniae accounts for 20 to 30% of all community-acquired pneumonia and has been associated with other airway pathologies, including asthma, and a range of extrapulmonary manifestations. although the entire genomic sequence of m. pneumoniae has been completed, the functions of many of these genes in mycoplasma physiology are unknown. in this study, we focused on clpb, a well-known heat shock gene in other bacteria, to examine its role in mycoplasma growth. transcriptional and transl ...200818779336
synthesis and characterization of new piperazine-type inhibitors for mitochondrial nadh-ubiquinone oxidoreductase (complex i).the mode of action of deltalac-acetogenins, strong inhibitors of bovine heart mitochondrial complex i, is different from that of traditional inhibitors such as rotenone and piericidin a [murai, m., et al. (2007) biochemistry 46 , 6409-6416]. as further exploration of these unique inhibitors might provide new insights into the terminal electron transfer step of complex i, we drastically modified the structure of deltalac-acetogenins and characterized their inhibitory action. in particular, on the ...200818781777
the putative rnase p motif in the dead box helicase hera is dispensable for efficient interaction with rna and helicase activity.dead box helicases use the energy of atp hydrolysis to remodel rna structures or rna/protein complexes. they share a common helicase core with conserved signature motifs, and additional domains may confer substrate specificity. identification of a specific substrate is crucial towards understanding the physiological role of a helicase. rna binding and atpase stimulation are necessary, but not sufficient criteria for a bona fide helicase substrate. here, we report single molecule fret experiments ...200818782831
the human mitochondrial ribosome recycling factor is essential for cell viability.the molecular mechanism of human mitochondrial translation has yet to be fully described. we are particularly interested in understanding the process of translational termination and ribosome recycling in the mitochondrion. several candidates have been implicated, for which subcellular localization and characterization have not been reported. here, we show that the putative mitochondrial recycling factor, mtrrf, is indeed a mitochondrial protein. expression of human mtrrf in fission yeast devoid ...200818782833
concurrent nucleation of 16s folding and induced fit in 30s ribosome assembly.rapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. how the escherichia coli 16s ribosomal rna and the 20 proteins that make up the 30s ribosomal subunit can assemble correctly in a few minutes remains a challenging problem, partly because of the lack of real-time data on the earliest stages of assembly. by providing snapshots of individual rna and protein interactions as they emerge in real time, here we show tha ...200818784650
insights into the mode of action of a putative zinc transporter czrb in thermus thermophilus.the crystal structures of the cytoplasmic domain of the putative zinc transporter czrb in the apo and zinc-bound forms reported herein are consistent with the protein functioning in vivo as a homodimer. nmr, x-ray scattering, and size-exclusion chromatography provide support for dimer formation. full-length variants of czrb in the apo and zinc-loaded states were generated by homology modeling with the zn2+/h+ antiporter yiip. the model suggests a way in which zinc binding to the cytoplasmic frag ...200818786400
mass spectrometry profiles superoxide-induced intramolecular disulfide in the fmn-binding subunit of mitochondrial complex i.protein thiols with regulatory functions play a critical role in maintaining the homeostasis of the redox state in mitochondria. one major host of regulatory cysteines in mitochondria is complex i, with the thiols primarily located on its 51 kda fmn-binding subunit. in response to oxidative stress, these thiols are expected to form intramolecular disulfide bridges as one of their oxidative post-translational modifications. here, to test this hypothesis and gain insights into the molecular patter ...200818789718
mycobacterium tuberculosis cyp130: crystal structure, biophysical characterization, and interactions with antifungal azole drugs.cyp130 is one of the 20 mycobacterium tuberculosis cytochrome p450 enzymes, only two of which, cyp51 and cyp121, have so far been studied as individually expressed proteins. here we characterize a third heterologously expressed m. tuberculosis cytochrome p450, cyp130, by uv-visible spectroscopy, isothermal titration calorimetry, and x-ray crystallography, including determination of the crystal structures of ligand-free and econazole-bound cyp130 at a resolution of 1.46 and 3.0a(,) respectively. ...200818089574
mycobacterium tuberculosis cyp130: crystal structure, biophysical characterization, and interactions with antifungal azole drugs.cyp130 is one of the 20 mycobacterium tuberculosis cytochrome p450 enzymes, only two of which, cyp51 and cyp121, have so far been studied as individually expressed proteins. here we characterize a third heterologously expressed m. tuberculosis cytochrome p450, cyp130, by uv-visible spectroscopy, isothermal titration calorimetry, and x-ray crystallography, including determination of the crystal structures of ligand-free and econazole-bound cyp130 at a resolution of 1.46 and 3.0a(,) respectively. ...200818089574
mammalian 2',3' cyclic nucleotide phosphodiesterase (cnp) can function as a trna splicing enzyme in vivo.yeast and plant trna splicing entails discrete healing and sealing steps catalyzed by a trna ligase that converts the 2',3' cyclic phosphate and 5'-oh termini of the broken trna exons to 3'-oh/2'-po4 and 5'-po4 ends, respectively, then joins the ends to yield a 2'-po4, 3'-5' phosphodiester splice junction. the junction 2'-po4 is removed by a trna phosphotransferase, tpt1. animal cells have two potential trna repair pathways: a yeast-like system plus a distinctive mechanism, also present in archa ...200818094118
assembly of the 5' and 3' minor domains of 16s ribosomal rna as monitored by tethered probing from ribosomal protein s20.the ribosomal protein (r-protein) s20 is a primary binding protein. as such, it interacts directly and independently with the 5' domain as well as the 3' minor domain of 16s ribosomal rna (rrna) in minimal particles and the fully assembled 30s subunit. the interactions observed between r-protein s20 and the 5' domain of 16s rrna are quite extensive, while those between r-protein s20 and the 3' minor domain are significantly more limited. in this study, directed hydroxyl radical probing mediated ...200818155048
assembly of the 5' and 3' minor domains of 16s ribosomal rna as monitored by tethered probing from ribosomal protein s20.the ribosomal protein (r-protein) s20 is a primary binding protein. as such, it interacts directly and independently with the 5' domain as well as the 3' minor domain of 16s ribosomal rna (rrna) in minimal particles and the fully assembled 30s subunit. the interactions observed between r-protein s20 and the 5' domain of 16s rrna are quite extensive, while those between r-protein s20 and the 3' minor domain are significantly more limited. in this study, directed hydroxyl radical probing mediated ...200818155048
a flexible peptide tether controls accessibility of a unique c-terminal rna-binding domain in leucyl-trna synthetases.a unique c-terminal domain extension is required by most leucyl-trna synthetases (leurs) for aminoacylation. in one exception, the enzymatic activity of yeast mitochondrial leurs is actually impeded by its own c-terminal domain. it was proposed that the yeast mitochondrial leurs has compromised its aminoacylation activity to some extent and adapted its c terminus for a second role in rna splicing, which is also essential. x-ray crystal structures of the leurs-trna complex show that the 60 residu ...200818155724
a flexible peptide tether controls accessibility of a unique c-terminal rna-binding domain in leucyl-trna synthetases.a unique c-terminal domain extension is required by most leucyl-trna synthetases (leurs) for aminoacylation. in one exception, the enzymatic activity of yeast mitochondrial leurs is actually impeded by its own c-terminal domain. it was proposed that the yeast mitochondrial leurs has compromised its aminoacylation activity to some extent and adapted its c terminus for a second role in rna splicing, which is also essential. x-ray crystal structures of the leurs-trna complex show that the 60 residu ...200818155724
crystal structure of bacillus stearothermophilus uvra provides insight into atp-modulated dimerization, uvrb interaction, and dna binding.the nucleotide excision repair pathway corrects many structurally unrelated dna lesions. damage recognition in bacteria is performed by uvra, a member of the abc atpase superfamily whose functional form is a dimer with four nucleotide-binding domains (nbds), two per protomer. in the 3.2 a structure of uvra from bacillus stearothermophilus, we observe that the nucleotide-binding sites are formed in an intramolecular fashion and are not at the dimer interface as is typically found in other abc atp ...200818158267
crystal structure of bacillus stearothermophilus uvra provides insight into atp-modulated dimerization, uvrb interaction, and dna binding.the nucleotide excision repair pathway corrects many structurally unrelated dna lesions. damage recognition in bacteria is performed by uvra, a member of the abc atpase superfamily whose functional form is a dimer with four nucleotide-binding domains (nbds), two per protomer. in the 3.2 a structure of uvra from bacillus stearothermophilus, we observe that the nucleotide-binding sites are formed in an intramolecular fashion and are not at the dimer interface as is typically found in other abc atp ...200818158267
protein factors in pre-mrna 3'-end processing.most eukaryotic mrna precursors (premrnas) must undergo extensive processing, including cleavage and polyadenylation at the 3'-end. processing at the 3'-end is controlled by sequence elements in the pre-mrna (cis elements) as well as protein factors. despite the seeming biochemical simplicity of the processing reactions, more than 14 proteins have been identified for the mammalian complex, and more than 20 proteins have been identified for the yeast complex. the 3'-end processing machinery also ...200818158581
akap18 contains a phosphoesterase domain that binds amp.protein kinase a anchoring proteins (akaps), defined by their capacity to target the camp-dependent protein kinase to distinct subcellular locations, function as molecular scaffolds mediating the assembly of multicomponent complexes to integrate and organise multiple signalling events. despite their central importance in regulating cellular processes, little is known regarding their diverse structures and molecular mechanisms. here, using bioinformatics and x-ray crystallography, we define a cen ...200818082768
akap18 contains a phosphoesterase domain that binds amp.protein kinase a anchoring proteins (akaps), defined by their capacity to target the camp-dependent protein kinase to distinct subcellular locations, function as molecular scaffolds mediating the assembly of multicomponent complexes to integrate and organise multiple signalling events. despite their central importance in regulating cellular processes, little is known regarding their diverse structures and molecular mechanisms. here, using bioinformatics and x-ray crystallography, we define a cen ...200818082768
escherichia coli cytosolic glycerophosphodiester phosphodiesterase (ugpq) requires mg2+, co2+, or mn2+ for its enzyme activity.escherichia coli cytosolic glycerophosphodiester phosphodiesterase, ugpq, functions in the absence of other proteins encoded by the ugp operon and requires mg2+, mn2+, or co2+, in contrast to ca2+-dependent periplasmic glycerophosphodiester phosphodiesterase, glpq. ugpq has broad substrate specificity toward various glycerophosphodiesters, producing sn-glycerol-3-phosphate and the corresponding alcohols. ugpq accumulates under conditions of phosphate starvation, suggesting that it allows the uti ...200818083802
escherichia coli cytosolic glycerophosphodiester phosphodiesterase (ugpq) requires mg2+, co2+, or mn2+ for its enzyme activity.escherichia coli cytosolic glycerophosphodiester phosphodiesterase, ugpq, functions in the absence of other proteins encoded by the ugp operon and requires mg2+, mn2+, or co2+, in contrast to ca2+-dependent periplasmic glycerophosphodiester phosphodiesterase, glpq. ugpq has broad substrate specificity toward various glycerophosphodiesters, producing sn-glycerol-3-phosphate and the corresponding alcohols. ugpq accumulates under conditions of phosphate starvation, suggesting that it allows the uti ...200818083802
structural basis for different substrate specificities of two adp-ribose pyrophosphatases from thermus thermophilus hb8.adp-ribose (adpr) is one of the main substrates of nudix proteins. among the eight nudix proteins of thermus thermophilus hb8, we previously determined the crystal structure of ndx4, an adpr pyrophosphatase (adprase). in this study we show that ndx2 of t. thermophilus also preferentially hydrolyzes adpr and flavin adenine dinucleotide and have determined its crystal structure. we have determined the structures of ndx2 alone and in complex with mg2+, with mg2+ and amp, and with mg2+ and a nonhydr ...200818039767
discrimination of class i cyclobutane pyrimidine dimer photolyase from blue light photoreceptors by single methionine residue.dna photolyase recognizes ultraviolet-damaged dna and breaks improperly formed covalent bonds within the cyclobutane pyrimidine dimer by a light-activated electron transfer reaction between the flavin adenine dinucleotide, the electron donor, and cyclobutane pyrimidine dimer, the electron acceptor. theoretical analysis of the electron-tunneling pathways of the dna photolyase derived from anacystis nidulans can reveal the active role of the protein environment in the electron transfer reaction. h ...200818055535
discrimination of class i cyclobutane pyrimidine dimer photolyase from blue light photoreceptors by single methionine residue.dna photolyase recognizes ultraviolet-damaged dna and breaks improperly formed covalent bonds within the cyclobutane pyrimidine dimer by a light-activated electron transfer reaction between the flavin adenine dinucleotide, the electron donor, and cyclobutane pyrimidine dimer, the electron acceptor. theoretical analysis of the electron-tunneling pathways of the dna photolyase derived from anacystis nidulans can reveal the active role of the protein environment in the electron transfer reaction. h ...200818055535
redox properties of thermus thermophilus ba3: different electron-proton coupling in oxygen reductases?a comprehensive study of the thermodynamic redox behavior of the hemes of the ba3 enzyme from thermus thermophilus, a b-type heme-copper oxygen reductase, is presented. this enzyme, in contrast to those having a single type of heme, allows the b- and a-type hemes to be monitored separately by visible spectroscopy and the reduction potential of each heme to be determined unequivocally. the relative order of the midpoint reduction potentials of each center changed in the ph range from 6 to 8.4, an ...200818065462
yeast as a model of human mitochondrial trna base substitutions: investigation of the molecular basis of respiratory defects.we investigate the relationships between acylation defects and structure alterations due to base substitutions in yeast mitochondrial (mt) trna(uur)(leu). the studied substitutions are equivalent to the a3243g and t3250c human pathogenetic trna mutations. our data show that both mutations can produce trna(uur)(leu) acylation defects, although to a different extent. for mutant a14g (equivalent to melas a3243g base substitution), the presence of the trna and its defective aminoacylation could be o ...200818065717
structural basis of a ribozyme's thermostability: p1-l9 interdomain interaction in rnase p rna.for stability, many catalytic rnas rely on long-range tertiary interactions, the precise role of each often being unclear. here we demonstrate that one of the three interdomain architectural struts of rnase p rna (p rna) is the key to activity at higher temperatures: disrupting the p1-l9 helix-tetraloop interaction in p rna of the thermophile thermus thermophilus decreased activity at high temperatures in the rna-alone reaction and at low mg2+ concentrations in the holoenzyme reaction. conversel ...200817998289
a novel chromatography system to isolate active ribosomes from pathogenic bacteria.we have developed a novel chromatography for the rapid isolation of active ribosomes from bacteria without the use of harsh conditions or lengthy procedures that damage ribosomes. ribosomes interact with an alkyl linker attached to the resin, apparently through their rna component. examples are given with ribosomes from escherichia coli, deinococcus radiodurans, and with clinical isolates of streptococcus pneumoniae and methicillin-resistant staphylococcus aureus (mrsa). the ribosomes obtained b ...200817998293
the process of displacing the single-stranded dna-binding protein from single-stranded dna by reco and recr proteins.the regions of single-stranded (ss) dna that result from dna damage are immediately coated by the ssdna-binding protein (ssb). recf pathway proteins facilitate the displacement of ssb from ssdna, allowing the reca protein to form protein filaments on the ssdna region, which facilitates the process of recombinational dna repair. in this study, we examined the mechanism of ssb displacement from ssdna using purified thermus thermophilus recf pathway proteins. to date, reco and recr are thought to a ...200818000001
eukaryotic ribosomal rna determinants of aminoglycoside resistance and their role in translational fidelity.recent studies of prokaryotic ribosomes have dramatically increased our knowledge of ribosomal rna (rrna) structure, functional centers, and their interactions with antibiotics. however, much less is known about how rrna function differs between prokaryotic and eukaryotic ribosomes. the core decoding sites are identical in yeast and human 18s rrnas, suggesting that insights obtained in studies with yeast rrna mutants can provide information about ribosome function in both species. in this study, ...200818003936
the methyltransferase yfgb/rlmn is responsible for modification of adenosine 2503 in 23s rrna.a2503 in 23s rrna of the gram-negative bacterium escherichia coli is located in a functionally important region of the ribosome, at the entrance to the nascent peptide exit tunnel. in e. coli, and likely in other species, this adenosine residue is post-transcriptionally modified to m2a. the enzyme responsible for this modification was previously unknown. we identified e. coli protein yfgb, which belongs to the radical sam enzyme superfamily, as the methyltransferase that modifies a2503 of 23s rr ...200818025251
the role of the mitochondrial glycine cleavage complex in the metabolism and virulence of the protozoan parasite leishmania major.for the human pathogen leishmania major, a key metabolic function is the synthesis of thymidylate, which requires 5,10-methylenetetrahydrofolate (5,10-ch(2)-thf). 5,10-ch(2)-thf can be synthesized from glycine by the mitochondrial glycine cleavage complex (gcc). bioinformatic analysis revealed the four subunits of the gcc in the l. major genome, and the role of the gcc in parasite metabolism and virulence was assessed through studies of the p subunit (glycine decarboxylase (gcvp)). first, a tagg ...200817981801
the role of the mitochondrial glycine cleavage complex in the metabolism and virulence of the protozoan parasite leishmania major.for the human pathogen leishmania major, a key metabolic function is the synthesis of thymidylate, which requires 5,10-methylenetetrahydrofolate (5,10-ch(2)-thf). 5,10-ch(2)-thf can be synthesized from glycine by the mitochondrial glycine cleavage complex (gcc). bioinformatic analysis revealed the four subunits of the gcc in the l. major genome, and the role of the gcc in parasite metabolism and virulence was assessed through studies of the p subunit (glycine decarboxylase (gcvp)). first, a tagg ...200817981801
the stator complex of the a1a0-atp synthase--structural characterization of the e and h subunits.archaeal atp synthase (a-atpase) is the functional homolog to the atp synthase found in bacteria, mitochondria and chloroplasts, but the enzyme is structurally more related to the proton-pumping vacuolar atpase found in the endomembrane system of eukaryotes. we have cloned, overexpressed and characterized the stator-forming subunits e and h of the a-atpase from the thermoacidophilic archaeon, thermoplasma acidophilum. size exclusion chromatography, cd, matrix-assisted laser desorption ionization ...200818036615
the stator complex of the a1a0-atp synthase--structural characterization of the e and h subunits.archaeal atp synthase (a-atpase) is the functional homolog to the atp synthase found in bacteria, mitochondria and chloroplasts, but the enzyme is structurally more related to the proton-pumping vacuolar atpase found in the endomembrane system of eukaryotes. we have cloned, overexpressed and characterized the stator-forming subunits e and h of the a-atpase from the thermoacidophilic archaeon, thermoplasma acidophilum. size exclusion chromatography, cd, matrix-assisted laser desorption ionization ...200818036615
common thiolation mechanism in the biosynthesis of trna thiouridine and sulphur-containing cofactors.2-thioribothymidine (s(2)t), a modified uridine, is found at position 54 in transfer rnas (trnas) from several thermophiles; s(2)t stabilizes the l-shaped structure of trna and is essential for growth at higher temperatures. here, we identified an atpase (trna-two-thiouridine c, ttuc) required for the 2-thiolation of s(2)t in thermus thermophilus and examined in vitro s(2)t formation by ttuc and previously identified s(2)t-biosynthetic proteins (ttua, ttub, and cysteine desulphurases). the c-ter ...200819037260
purification, crystallization and x-ray structures of the two manganese superoxide dismutases from caenorhabditis elegans.caenorhabditis elegans expresses two manganese superoxide dismutase enzymes (mnsod-2 and mnsod-3) that are targeted to the mitochondrion. mnsod-2 is constitutively expressed, while synthesis of mnsod-3 is inducible. the structures of these two mononuclear metalloenzymes have been determined to 1.8 and 1.7 a resolution, respectively. pink crystals formed in space group p4(1)2(1)2 for each, with unit-cell parameters a = b = 81.0, c = 137.4 a for mnsod-2 and a = b = 81.8, c = 136.0 a for mnsod-3. t ...200819052361
cloning, expression, crystallization and preliminary x-ray crystallographic analysis of 3-dehydroquinate synthase, xoo1243, from xanthomonas oryzae pv. oryzae.the disease bacterial blight results in serious production losses of rice in asian countries. the arob gene encoding dehydroquinate synthase (dhqs), which is a potential antibiotic target, was identified from the plant-pathogenic bacterium xanthomonas oryzae pv. oryzae (xoo). dhqs plays an essential role in the synthesis of aromatic compounds in the shikimate pathway. the arob gene (xoo1243) was cloned from xoo and the corresponding dhqs protein was subsequently overexpressed in escherichia coli ...200819052366
crystallization and preliminary x-ray diffraction studies of the prototypal homologue of mitoneet (tth-neet0026) from the extreme thermophile thermus thermophilus hb8.mitoneet (a mammalian mitochondrial outer membrane protein) is a potential pharmacological and clinical target of the insulin-sensitizer pioglitazone. the thermophilic homologue of mitoneet (ttha0026) from thermus thermophilus hb8 has been heterologously overproduced in escherichia coli and purified as a water-soluble prototypal protein containing the mitoneet-like [2fe-2s] cluster. the resultant recombinant protein, named tth-neet0026, has been crystallized in its oxidized form by the hanging-d ...200819052371
the 1.6 a crystal structure of mycobacterium smegmatis mshc: the penultimate enzyme in the mycothiol biosynthetic pathway.mycobacterium smegmatis mshc catalyzes the atp-dependent condensation of glcn-ins and l-cysteine to form l-cys-glcn-ins, the penultimate step in mycothiol biosynthesis. attempts to crystallize the native, full-length mshc have been unsuccessful. however, incubation of the enzyme with the cysteinyl adenylate analogue, 5'-o-[n-(l-cysteinyl)-sulfamonyl]adenosine (csa), followed by a 24-h limited trypsin proteolysis yielded an enzyme preparation that readily crystallized. the three-dimensional struc ...200819053270
recombinant production and biochemical characterization of a hyperthermostable alpha-glucan/maltodextrin phosphorylase from pyrococcus furiosus.alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-d-glucose-1-phosphate. we report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (pf1535) from a hyperthermophilic archaeon, pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. the apparent 98 kda recombinant enzyme was active over a broad range of temperatures and ...200819054743
recombinant production and biochemical characterization of a hyperthermostable alpha-glucan/maltodextrin phosphorylase from pyrococcus furiosus.alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-d-glucose-1-phosphate. we report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (pf1535) from a hyperthermophilic archaeon, pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. the apparent 98 kda recombinant enzyme was active over a broad range of temperatures and ...200819054743
bridge helix and trigger loop perturbations generate superactive rna polymerases.cellular rna polymerases are highly conserved enzymes that undergo complex conformational changes to coordinate the processing of nucleic acid substrates through the active site. two domains in particular, the bridge helix and the trigger loop, play a key role in this mechanism by adopting different conformations at various stages of the nucleotide addition cycle. the functional relevance of these structural changes has been difficult to assess from the relatively small number of static crystal ...200819055851
crystal structure of a translation termination complex formed with release factor rf2.we report the crystal structure of a translation termination complex formed by the thermus thermophilus 70s ribosome bound with release factor rf2, in response to a uaa stop codon, solved at 3 a resolution. the backbone of helix alpha5 and the side chain of serine of the conserved spf motif of rf2 recognize u1 and a2 of the stop codon, respectively. a3 is unstacked from the first 2 bases, contacting thr-216 and val-203 of rf2 and stacking on g530 of 16s rrna. the structure of the rf2 complex sup ...200819064930
vectorial proton transfer coupled to reduction of o2 and no by a heme-copper oxidase.the heme-copper oxidase (hcuo) superfamily consists of integral membrane proteins that catalyze the reduction of either oxygen or nitric oxide. the hcuos that reduce o(2) to h(2)o couple this reaction to the generation of a transmembrane proton gradient by using electrons and protons from opposite sides of the membrane and by pumping protons from inside the cell or organelle to the outside. the bacterial no-reductases (nor) reduce no to n(2)o (2no + 2e(-) + 2h(+) --> n(2)o + h(2)o), a reaction a ...200819074284
structure and functional role of dynein's microtubule-binding domain.dynein motors move various cargos along microtubules within the cytoplasm and power the beating of cilia and flagella. an unusual feature of dynein is that its microtubule-binding domain (mtbd) is separated from its ring-shaped aaa+ adenosine triphosphatase (atpase) domain by a 15-nanometer coiled-coil stalk. we report the crystal structure of the mouse cytoplasmic dynein mtbd and a portion of the coiled coil, which supports a mechanism by which the atpase domain and mtbd may communicate through ...200819074350
heterogeneity of large macromolecular complexes revealed by 3d cryo-em variance analysis.macromolecular structure determination by cryo-electron microscopy (em) and single-particle analysis are based on the assumption that imaged molecules have identical structure. with the increased size of processed data sets, it becomes apparent that many complexes coexist in a mixture of conformational states or contain flexible regions. we describe an implementation of the bootstrap resampling technique that yields estimates of voxel-by-voxel variance of a structure reconstructed from the set o ...200819081053
structural basis for inactivation of the human pyruvate dehydrogenase complex by phosphorylation: role of disordered phosphorylation loops.we report the crystal structures of the phosporylated pyruvate dehydrogenase (e1p) component of the human pyruvate dehydrogenase complex (pdc). the complete phosphorylation at ser264-alpha (site 1) of a variant e1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the pdc core. we show that unlike its unmodified counterpart, the presence of a phosphoryl group at ser264-alpha prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three ph ...200819081061
revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein l22.bacterial antibiotic resistance can occur by many mechanisms. an intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. for example, a 3-residue deletion (deltamkr) in ribosomal protein l22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. here, however, we demonstrate that deltamkr and wild-type ribosomes show comparab ...200819015512
transposition of an insertion sequence, istth7, in the genome of the extreme thermophile thermus thermophilus hb8.we have identified an active insertion sequence (is) in the genome of thermus thermophilus hb8. transposition was detected as insertional inactivation of a 16s rrna methyltransferase gene, rsmg, resulting in streptomycin resistance. the is element, istth7, is 1029 bp in length, encodes an imperfect 12 bp inverted repeat, and produces a 9 bp direct repeat of the target sequence. the sequence of a putative transposase encoded by istth7 indicates that it is a member of the is427 group within the is ...200819016874
functional role of coenzyme q in the energy coupling of nadh-coq oxidoreductase (complex i): stabilization of the semiquinone state with the application of inside-positive membrane potential to proteoliposomes.coenzyme q10 (which is also designated as coq10, ubiquinone-10, uq10, coq, uq or simply as q) plays an important role in energy metabolism. for nadh-q oxidoreductase (complex i), ohnishi and salerno proposed a hypothesis that the proton pump is operated by the redox-driven conformational change of a q-binding protein, and that the bound form of semiquinone (sq) serves as its gate [febs letters 579 (2005) 45-55]. this was based on the following experimental results: (i) epr signals of the fast-re ...200819096096
structure of an argonaute silencing complex with a seed-containing guide dna and target rna duplex.here we report on a 3.0 a crystal structure of a ternary complex of wild-type thermus thermophilus argonaute bound to a 5'-phosphorylated 21-nucleotide guide dna and a 20-nucleotide target rna containing cleavage-preventing mismatches at the 10-11 step. the seed segment (positions 2 to 8) adopts an a-helical-like watson-crick paired duplex, with both ends of the guide strand anchored in the complex. an arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in a ...200819092929
urm1 at the crossroad of modifications. 'protein modifications: beyond the usual suspects' review series.the ubiquitin-like protein urm1 can be covalently conjugated to other proteins, such as the yeast thioredoxin peroxidase protein ahp1p, through a mechanism involving the ubiquitin e1-like enzyme uba4. recent findings have revealed a second function of urm1 as a sulphur carrier in the thiolation of eukaryotic cytoplasmic transfer rnas (trnas). interestingly, this new role of urm1 is similar to the sulphur-carrier activity of its prokaryotic counterparts, strengthening the hypothesis that urm1 is ...200819047990
function-biased choice of additives for optimization of protein crystallization - the case of the putative thioesterase pa5185 from pseudomonas aeruginosa pao1.the crystal structure of pa5185, a putative thioesterase from pseudomonas aeruginosa strain pao1, was solved using multi-wavelength anomalous diffraction to 2.4 a. analysis of the structure and information about the putative function of the protein were used to optimize crystallization conditions. the crystal growth was optimized by applying additives with chemical similarity to a fragment of a putative pa5185 substrate (coa or its derivative). using new crystallization conditions containing thi ...200819898606
large deviations for random trees and the branching of rna secondary structures.we give a large deviation principle (ldp) with explicit rate function for the distribution of vertex degrees in plane trees, a combinatorial model of rna secondary structures. we calculate the typical degree distributions based on nearest neighbor free energies, and compare our results with the branching configurations found in two sets of large rna secondary structures. we find substantial agreement overall, with some interesting deviations which merit further study.200819083065
large deviations for random trees and the branching of rna secondary structures.we give a large deviation principle (ldp) with explicit rate function for the distribution of vertex degrees in plane trees, a combinatorial model of rna secondary structures. we calculate the typical degree distributions based on nearest neighbor free energies, and compare our results with the branching configurations found in two sets of large rna secondary structures. we find substantial agreement overall, with some interesting deviations which merit further study.200819083065
genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world.the first bacterial genome was sequenced in 1995, and the first archaeal genome in 1996. soon after these breakthroughs, an exponential rate of genome sequencing was established, with a doubling time of approximately 20 months for bacteria and approximately 34 months for archaea. comparative analysis of the hundreds of sequenced bacterial and dozens of archaeal genomes leads to several generalizations on the principles of genome organization and evolution. a crucial finding that enables function ...200818948295
minimum contradiction matrices in whole genome phylogenies.minimum contradiction matrices are a useful complement to distance-based phylogenies. a minimum contradiction matrix represents phylogenetic information under the form of an ordered distance matrix y(i) (,) (j) (n). a matrix element corresponds to the distance from a reference vertex n to the path (i, j). for an x-tree or a split network, the minimum contradiction matrix is a robinson matrix. it therefore fulfills all the inequalities defining perfect order: y(i) (,) (j) (n) >or= y(i) (,) (k) (n ...200819204821
evolutionary primacy of sodium bioenergetics.the f- and v-type atpases are rotary molecular machines that couple translocation of protons or sodium ions across the membrane to the synthesis or hydrolysis of atp. both the f-type (found in most bacteria and eukaryotic mitochondria and chloroplasts) and v-type (found in archaea, some bacteria, and eukaryotic vacuoles) atpases can translocate either protons or sodium ions. the prevalent proton-dependent atpases are generally viewed as the primary form of the enzyme whereas the sodium-transloca ...200818380897
bacterial heme-transport proteins and their heme-coordination modes.efficient iron acquisition is critical for an invading microbe's survival and virulence. most of the iron in mammals is incorporated into heme, which can be plundered by certain bacterial pathogens as a nutritional iron source. utilization of exogenous heme by bacteria involves the binding of heme or hemoproteins to the cell surface receptors, followed by the transport of heme into cells. once taken into the cytosol, heme is presented to heme oxygenases where the tetrapyrrole ring is cleaved in ...200818977196
bacterial heme-transport proteins and their heme-coordination modes.efficient iron acquisition is critical for an invading microbe's survival and virulence. most of the iron in mammals is incorporated into heme, which can be plundered by certain bacterial pathogens as a nutritional iron source. utilization of exogenous heme by bacteria involves the binding of heme or hemoproteins to the cell surface receptors, followed by the transport of heme into cells. once taken into the cytosol, heme is presented to heme oxygenases where the tetrapyrrole ring is cleaved in ...200818977196
the elusive object of desire--interactions of bacteriophages and their hosts.bacteria and their viruses (phages) are locked in an evolutionary contest, with each side producing constantly changing mechanisms of attack and defense that are aimed to increase the odds of survival. as a result, phages play central roles in a great variety of genetic processes and increase the rate of evolutionary change of the bacterial host, which could ultimately work to the benefit of the host in a long run.200818400552
structural biology of proline catabolism.the proline catabolic enzymes proline dehydrogenase and delta(1)-pyrroline-5-carboxylate dehydrogenase catalyze the 4-electron oxidation of proline to glutamate. these enzymes play important roles in cellular redox control, superoxide generation, apoptosis and cancer. in some bacteria, the two enzymes are fused into the bifunctional enzyme, proline utilization a. here we review the three-dimensional structural information that is currently available for proline catabolic enzymes. crystal structu ...200818369526
the application of fast-nmr for the identification of novel drug discovery targets.the continued success of genome sequencing projects has resulted in a wealth of information, but 40-50% of identified genes correspond to hypothetical proteins or proteins of unknown function. the functional annotation screening technology by nmr (fast-nmr) screen was developed to assign a biological function for these unannotated proteins with a structure solved by the protein structure initiative. fast-nmr is based on the premise that a biological function can be described by a similarity in b ...200818275915
soda: an mn/fe superoxide dismutase prediction and design server.superoxide dismutases (sods) are ubiquitous metalloenzymes that play an important role in the defense of aerobic organisms against oxidative stress, by converting reactive oxygen species into nontoxic molecules. we focus here on the sod family that uses fe or mn as cofactor.200818518943
tie me up, tie me down: inhibiting rna polymerase.mechanistic understanding of antibiotic action can yield crucial insights that aid in the design of new antibiotics. in this issue, mukhopadhyay et al. (2008) uncover the mechanism by which the antibiotic myxopyronin inhibits bacterial rna polymerase, suggesting a new target region in rna polymerase for inhibitor design.200818957193
cytochrome c oxidase: exciting progress and remaining mysteries.cytochrome c oxidase generates a proton motive force by two separate mechanisms. the first mechanism is similar to that postulated by peter mitchell, and is based on electrons and protons used to generate water coming from opposite sides of the membrane. the second mechanism was not initially anticipated, but is now firmly established as a proton pump. a brief review of the current state of our understanding of the proton pump of cytochrome oxidase is presented. we have come a long way since the ...200818975062
the chemistry and biochemistry of heme c: functional bases for covalent attachment.a discussion of the literature concerning the synthesis, function, and activity of heme c-containing proteins is presented. comparison of the properties of heme c, which is covalently bound to protein, is made to heme b, which is bound noncovalently. a question of interest is why nature uses biochemically expensive heme c in many proteins when its properties are expected to be similar to heme b. considering the effects of covalent heme attachment on heme conformation and on the proximal histidin ...200819030605
one heme, diverse functions: using biosynthetic myoglobin models to gain insights into heme-copper oxidases and nitric oxide reductases. 200818729107
the q-cycle reviewed: how well does a monomeric mechanism of the bc(1) complex account for the function of a dimeric complex?recent progress in understanding the q-cycle mechanism of the bc(1) complex is reviewed. the data strongly support a mechanism in which the q(o)-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron-sulfur protein is the rate-determining step for the overall process. the reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2fe-2s] cluster, in which the unfavorable protoni ...200818501698
nadh/nad+ interaction with nadh: ubiquinone oxidoreductase (complex i).the quantitative data on the binding affinity of nadh, nad(+), and their analogues for complex i as emerged from the steady-state kinetics data and from more direct studies under equilibrium conditions are summarized and discussed. the redox-dependency of the nucleotide binding and the reductant-induced change of fmn affinity to its tight non-covalent binding site indicate that binding (dissociation) of the substrate (product) may energetically contribute to the proton-translocating activity of ...200818471432
recognition of aminoacyl-trna: a common molecular mechanism revealed by cryo-em.the accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by ef-tu, which forms a ternary complex with aminoacyl(aa)-trna. to study the binding modes of different aa-trnas, we compared cryo-em maps of the kirromycin-stalled ribosome bound with ternary complexes containing phe-trna(phe), trp-trna(trp), or leu-trna(leui). the three maps suggest a common binding manner of cognate aa-trnas in their specific binding with both the ribosome and ef-tu. a ...200819020518
organization of an activator-bound rna polymerase holoenzyme.transcription initiation involves the conversion from closed promoter complexes, comprising rna polymerase (rnap) and double-stranded promoter dna, to open complexes, in which the enzyme is able to access the dna template in a single-stranded form. the complex between bacterial rnap and its major variant sigma factor sigma(54) remains as a closed complex until atp hydrolysis-dependent remodeling by activator proteins occurs. this remodeling facilitates dna melting and allows the transition to th ...200818995832
rifamycins do not function by allosteric modulation of binding of mg2+ to the rna polymerase active center.rifamycin antibacterial agents inhibit bacterial rna polymerase (rnap) by binding to a site adjacent to the rnap active center and preventing synthesis of rna products >2-3 nt in length. recently, artsimovitch et al. [(2005) cell 122:351-363] proposed that rifamycins function by allosteric modulation of binding of mg(2+) to the rnap active center and presented three lines of biochemical evidence consistent with this proposal. here, we show that rifamycins do not affect the affinity of binding of ...200818787125
enzymes used in molecular biology: a useful guide.since molecular cloning has become routine laboratory technique, manufacturers offer countless sources of enzymes to generate and manipulate nucleic acids. thus, selecting the appropriate enzyme for a specific task may seem difficult to the novice. this review aims at providing the readers with some cues for understanding the function and specificities of the different sources of polymerases, ligases, nucleases, phosphatases, methylases, and topoisomerases used for molecular cloning. we provide ...200818766469
advances in bacterial promoter recognition and its control by factors that do not bind dna.early work identified two promoter regions, the -10 and -35 elements, that interact sequence specifically with bacterial rna polymerase (rnap). however, we now know that several additional promoter elements contact rnap and influence transcription initiation. furthermore, our picture of promoter control has evolved beyond one in which regulation results solely from activators and repressors that bind to dna sequences near the rnap binding site: many important transcription factors bind directly ...200818521075
on helicases and other motor proteins.helicases are molecular machines that utilize energy derived from atp hydrolysis to move along nucleic acids and to separate base-paired nucleotides. the movement of the helicase can also be described as a stationary helicase that pumps nucleic acid. recent structural data for the hexameric e1 helicase of papillomavirus in complex with single-stranded dna and mgadp has provided a detailed atomic and mechanistic picture of its atp-driven dna translocation. the structural and mechanistic features ...200818329872
"hot cores" in proteins: comparative analysis of the apolar contact area in structures from hyper/thermophilic and mesophilic organisms.a wide variety of stabilizing factors have been invoked so far to elucidate the structural basis of protein thermostability. these include, amongst the others, a higher number of ion-pairs interactions and hydrogen bonds, together with a better packing of hydrophobic residues. it has been frequently observed that packing of hydrophobic side chains is improved in hyperthermophilic proteins, when compared to their mesophilic counterparts. in this work, protein crystal structures from hyper/thermop ...200818312638
macromolecular micromovements: how rna polymerase translocates.multi-subunit dna-dependent rna polymerases synthesize rna molecules thousands of nucleotides long. the reiterative reaction of nucleotide condensation occurs at rates of tens of nucleotides per second, invariably linked to the translocation of the enzyme along the dna template, or threading of the dna and the nascent rna molecule through the enzyme. reiteration of the nucleotide addition/translocation cycle without dissociation from the dna and rna requires both isomorphic and metamorphic confo ...200919889534
elongation in translation as a dynamic interaction among the ribosome, trna, and elongation factors ef-g and ef-tu.the ribosome is a complex macromolecular machine that translates the message encoded in the messenger rna and synthesizes polypeptides by linking the individual amino acids carried by the cognate transfer rnas (trnas). the protein elongation cycle, during which the trnas traverse the ribosome in a coordinated manner along a path of more than 100 a, is facilitated by large-scale rearrangements of the ribosome. these rearrangements go hand in hand with conformational changes of trna as well as elo ...200920025795
hydrogen bonding and packing density are factors most strongly connected to limiting sites of high flexibility in the 16s rrna in the 30s ribosome.conformational flexibility in structured rna frequently is critical to function. the 30s ribosomal subunit exists in different conformations in different functional states due to changes in the central part of the 16s rrna. we are interested in evaluating the factors that might be responsible for restricting flexibility to specific parts of the 16s rrna using biochemical data obtained from the 30s subunit in solution. this problem was approached taking advantage of the observation that there mus ...200919643000
rna polymerase active center: the molecular engine of transcription.rna polymerase (rnap) is a complex molecular machine that governs gene expression and its regulation in all cellular organisms. to accomplish its function of accurately producing a full-length rna copy of a gene, rnap performs a plethora of chemical reactions and undergoes multiple conformational changes in response to cellular conditions. at the heart of this machine is the active center, the engine, which is composed of distinct fixed and moving parts that serve as the ultimate acceptor of reg ...200919489723
electron flow through proteins.electron transfers in photosynthesis and respiration commonly occur between metal-containing cofactors that are separated by large molecular distances. employing laser flash-quench triggering methods, we have shown that 20-å, coupling-limited fe(ii) to ru(iii) and cu(i) to ru(iii) electron tunneling in ru-modified cytochromes and blue copper proteins can occur on the microsecond timescale both in solutions and crystals. redox equivalents can be transferred even longer distances by multistep tunn ...200920161522
a stable hyponitrite-bridged iron porphyrin complex.the coupling of two nitric oxide (no) molecules in heme active sites is an important contributor to the conversion of no to nitrous oxide (n(2)o) by heme-containing enzymes. several formulations for the presumed heme-fe{n(2)o(2)}(n-) intermediates have been proposed previously, however, no crystal structures of heme-fe{n(2)o(2)}(n-) systems have been reported to date. we report the first isolation and characterization of a stable bimetallic hyponitrite iron porphyrin, [(oep)fe](2)(mu-n(2)o(2)), ...200919191487
architecture of complex i and its implications for electron transfer and proton pumping.proton pumping nadh:ubiquinone oxidoreductase (complex i) is the largest and remains by far the least understood enzyme complex of the respiratory chain. it consists of a peripheral arm harbouring all known redox active prosthetic groups and a membrane arm with a yet unknown number of proton translocation sites. the ubiquinone reduction site close to iron-sulfur cluster n2 at the interface of the 49-kda and psst subunits has been mapped by extensive site directed mutagenesis. independent lines o ...200919366614
molecular evolution of multisubunit rna polymerases: sequence analysis.transcription in all cellular organisms is performed by multisubunit, dna-dependent rna polymerases that synthesize rna from dna templates. previous sequence and structural studies have elucidated the importance of shared regions common to all multisubunit rna polymerases. in addition, rna polymerases contain multiple lineage-specific domain insertions involved in protein-protein and protein-nucleic acid interactions. we have created comprehensive multiple sequence alignments using all available ...200919895820
molecular evolution of multisubunit rna polymerases: sequence analysis.transcription in all cellular organisms is performed by multisubunit, dna-dependent rna polymerases that synthesize rna from dna templates. previous sequence and structural studies have elucidated the importance of shared regions common to all multisubunit rna polymerases. in addition, rna polymerases contain multiple lineage-specific domain insertions involved in protein-protein and protein-nucleic acid interactions. we have created comprehensive multiple sequence alignments using all available ...200919895820
comphy: prokaryotic composite distance phylogenies inferred from whole-genome gene sets.with the increasing availability of whole genome sequences, it is becoming more and more important to use complete genome sequences for inferring species phylogenies. we developed a new tool comphy, 'composite distance phylogeny', based on a composite distance matrix calculated from the comparison of complete gene sets between genome pairs to produce a prokaryotic phylogeny.200919208152
assessing the quality of whole genome alignments in bacteria.comparing genomes is an essential preliminary step to solve many problems in biology. matching long similar segments between two genomes is a precondition for their evolutionary, genetic, and genome rearrangement analyses. though various comparison methods have been developed in recent years, a quantitative assessment of their performance is lacking. here, we describe two families of assessment measures whose purpose is to evaluate bacteria-oriented comparison tools. the first measure is based o ...200920049164
a kernel for open source drug discovery in tropical diseases.conventional patent-based drug development incentives work badly for the developing world, where commercial markets are usually small to non-existent. for this reason, the past decade has seen extensive experimentation with alternative r&d institutions ranging from private-public partnerships to development prizes. despite extensive discussion, however, one of the most promising avenues-open source drug discovery-has remained elusive. we argue that the stumbling block has been the absence of a c ...200919381286
evolution of prokaryotic spfh proteins.the spfh protein superfamily is a diverse family of proteins whose eukaryotic members are involved in the scaffolding of detergent-resistant microdomains. recently the origin of the spfh proteins has been questioned. instead, convergent evolution has been proposed. however, an independent, convergent evolution of three large prokaryotic and three eukaryotic families is highly unlikely, especially when other mechanisms such as lateral gene transfer which could also explain their distribution patt ...200919138386
amidoligases with atp-grasp, glutamine synthetase-like and acetyltransferase-like domains: synthesis of novel metabolites and peptide modifications of proteins.recent studies have shown that the ubiquitin system had its origins in ancient cofactor/amino acid biosynthesis pathways. preliminary studies also indicated that conjugation systems for other peptide tags on proteins, such as pupylation, have evolutionary links to cofactor/amino acid biosynthesis pathways. following up on these observations, we systematically investigated the non-ribosomal amidoligases of the atp-grasp, glutamine synthetase-like and acetyltransferase folds by classifying the kno ...200920023723
target selection and annotation for the structural genomics of the amidohydrolase and enolase superfamilies.to study the substrate specificity of enzymes, we use the amidohydrolase and enolase superfamilies as model systems; members of these superfamilies share a common tim barrel fold and catalyze a wide range of chemical reactions. here, we describe a collaboration between the enzyme specificity consortium (enspec) and the new york sgx research center for structural genomics (nysgxrc) that aims to maximize the structural coverage of the amidohydrolase and enolase superfamilies. using sequence- and s ...200919219566
horizontal gene transfer of zinc and non-zinc forms of bacterial ribosomal protein s4.the universal ribosomal protein s4 is essential for the initiation of small subunit ribosomal assembly and translational accuracy. being part of the information processing machinery of the cell, the gene for s4 is generally thought of as being inherited vertically and has been used in concatenated gene phylogenies. here we report the evolution of ribosomal protein s4 in relation to a broad sharing of zinc/non-zinc forms of the gene and study the scope of horizontal gene transfer (hgt) of s4 duri ...200919640295
posttranslational control of transcription factor fixk2, a key regulator for the bradyrhizobium japonicum-soybean symbiosis.rhizobial fixk-like proteins play essential roles in activating genes for endosymbiotic life in legume root nodules, such as genes for micro-oxic respiration. in the facultative soybean symbiont, bradyrhizobium japonicum, the fixk(2) protein is the key player in a complex regulatory network. the fixk(2) gene itself is activated by the 2-component regulatory system fixlj in response to a moderate decrease of the oxygen tension, and the fixk(2) protein distributes and amplifies this response to th ...200919955406
the crystal structure of apo-ftsh reveals domain movements necessary for substrate unfolding and translocation.the hexameric membrane-spanning atp-dependent metalloprotease ftsh is universally conserved in eubacteria, mitochondria, and chloroplasts, where it fulfills key functions in quality control and signaling. as a member of the self-compartmentalizing atpases associated with various cellular activities (aaa+ proteases), ftsh converts the chemical energy stored in atp via conformational rearrangements into a mechanical force that is used for substrate unfolding and translocation into the proteolytic ...200919955424
tissue inhibitor of metalloproteinase 1 expression associated with gene demethylation confers anoikis resistance in early phases of melanocyte malignant transformation.although anoikis resistance has been considered a hallmark of malignant phenotype, the causal relation between neoplastic transformation and anchorage-independent growth remains undefined. we developed an experimental model of murine melanocyte malignant transformation, where a melanocyte lineage (melan-a) was submitted to sequential cycles of anchorage blockade, resulting in progressive morphologic alterations, and malignant transformation. throughout this process, cells corresponding to premal ...200919956395
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