| crystallization and preliminary x-ray analysis of a native human trna synthetase whose allelic variants are associated with charcot-marie-tooth disease. | glycyl-trna synthetase (glyrs) is one of a group of enzymes that catalyze the synthesis of aminoacyl-trnas for translation. mutations of human and mouse glyrss are causally associated with charcot-marie-tooth disease, the most common genetic disorder of the peripheral nervous system. as the first step towards a structure-function analysis of this disease, native human glyrs was expressed, purified and crystallized. the crystal belonged to space group p4(3)2(1)2 or its enantiomorphic space group ... | 2006 | 17142907 |
| biochemical and structural characterization of the secreted chorismate mutase (rv1885c) from mycobacterium tuberculosis h37rv: an *aroq enzyme not regulated by the aromatic amino acids. | the gene rv1885c from the genome of mycobacterium tuberculosis h37rv encodes a monofunctional and secreted chorismate mutase (*mtcm) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the *aroq class of chorismate mutases. consistent with the heterologously expressed *mtcm having periplasmic destination in escherichia coli and the absence of a discrete periplasmic compartment in m. tuberculosis, we show here that *mtcm secretes into the culture filtrate of m. tuberculosis. *mtc ... | 2006 | 17146044 |
| the role of specific 2'-hydroxyl groups in the stabilization of the folded conformation of kink-turn rna. | the role of 2'-hydroxyl groups in stabilizing the tightly kinked geometry of the kink-turn (k-turn) has been investigated. individual 2'-oh groups have been removed by chemical synthesis, and the kinking of the rna has been studied by gel electrophoresis and fluorescence resonance energy transfer. the results have been analyzed by reference to a database of 11 different crystallographic structures of k-turns. the potential hydrogen bonds fall into several classes. the most important are those in ... | 2007 | 17158708 |
| infantile encephalopathy and defective mitochondrial dna translation in patients with mutations of mitochondrial elongation factors efg1 and eftu. | mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial dna (mtdna)-encoded rnas and nuclear dna-encoded proteins. although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in rna-encoding mtdna genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. genetic investigation involving patient ... | 2007 | 17160893 |
| infantile encephalopathy and defective mitochondrial dna translation in patients with mutations of mitochondrial elongation factors efg1 and eftu. | mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial dna (mtdna)-encoded rnas and nuclear dna-encoded proteins. although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in rna-encoding mtdna genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. genetic investigation involving patient ... | 2007 | 17160893 |
| high precision multi-genome scale reannotation of enzyme function by eficaz. | the functional annotation of most genes in newly sequenced genomes is inferred from similarity to previously characterized sequences, an annotation strategy that often leads to erroneous assignments. we have performed a reannotation of 245 genomes using an updated version of eficaz, a highly precise method for enzyme function prediction. | 2006 | 17166279 |
| loss of a universal trna feature. | trna(his) has thus far always been found with one of the most distinctive of trna features, an extra 5' nucleotide that is usually a guanylate. trna(his) genes in a disjoint alphaproteobacterial group comprising the rhizobiales, rhodobacterales, caulobacterales, parvularculales, and pelagibacter generally fail to encode this extra guanylate, unlike those of other alphaproteobacteria and bacteria in general. rather than adding an extra 5' guanylate posttranscriptionally as eukaryotes do, evidence ... | 2007 | 17172343 |
| loss of a universal trna feature. | trna(his) has thus far always been found with one of the most distinctive of trna features, an extra 5' nucleotide that is usually a guanylate. trna(his) genes in a disjoint alphaproteobacterial group comprising the rhizobiales, rhodobacterales, caulobacterales, parvularculales, and pelagibacter generally fail to encode this extra guanylate, unlike those of other alphaproteobacteria and bacteria in general. rather than adding an extra 5' guanylate posttranscriptionally as eukaryotes do, evidence ... | 2007 | 17172343 |
| x-ray crystal structure of mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase ii (mtkasb). | mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the mycobacterium tuberculosis cell wall. through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. mycobacteria possess two fatty acid synthases (fas); fas-i carries out de novo synthesis of fatty acids while fas-ii is considered to elongate medium chain length fatty acyl primers ... | 2007 | 17174327 |
| x-ray crystal structure of mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase ii (mtkasb). | mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the mycobacterium tuberculosis cell wall. through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. mycobacteria possess two fatty acid synthases (fas); fas-i carries out de novo synthesis of fatty acids while fas-ii is considered to elongate medium chain length fatty acyl primers ... | 2007 | 17174327 |
| evolutionary migration of a post-translationally modified active-site residue in the proton-pumping heme-copper oxygen reductases. | in the respiratory chains of aerobic organisms, oxygen reductase members of the heme-copper superfamily couple the reduction of o2 to proton pumping, generating an electrochemical gradient. there are three distinct families of heme-copper oxygen reductases: a, b, and c types. the a- and b-type oxygen reductases have an active-site tyrosine that forms a unique cross-linked histidine-tyrosine cofactor. in the c-type oxygen reductases (also called cbb3 oxidases), an analogous active-site tyrosine h ... | 2006 | 17176062 |
| scaffolding as an organizing principle in trans-translation. the roles of small protein b and ribosomal protein s1. | a eubacterial ribosome stalled on a defective mrna can be released through a quality control mechanism referred to as trans-translation, which depends on the coordinating binding actions of transfer-messenger rna, small protein b, and ribosome protein s1. by means of cryo-electron microscopy, we obtained a map of the complex composed of a stalled ribosome and small protein b, which appears near the decoding center. this result suggests that, when lacking a codon, the a-site on the small subunit ... | 2007 | 17179154 |
| scaffolding as an organizing principle in trans-translation. the roles of small protein b and ribosomal protein s1. | a eubacterial ribosome stalled on a defective mrna can be released through a quality control mechanism referred to as trans-translation, which depends on the coordinating binding actions of transfer-messenger rna, small protein b, and ribosome protein s1. by means of cryo-electron microscopy, we obtained a map of the complex composed of a stalled ribosome and small protein b, which appears near the decoding center. this result suggests that, when lacking a codon, the a-site on the small subunit ... | 2007 | 17179154 |
| functional organization of the rpb5 subunit shared by the three yeast rna polymerases. | rpb5, a subunit shared by the three yeast rna polymerases, combines a eukaryotic n-terminal module with a globular c-end conserved in all non-bacterial enzymes. conditional and lethal mutants of the moderately conserved eukaryotic module showed that its large n-terminal helix and a short motif at the end of the module are critical in vivo. lethal or conditional mutants of the c-terminal globe altered the binding of rpb5 to rpb1-beta25/26 (prolonging the bridge helix) and rpb1-alpha44/47 (ahead o ... | 2007 | 17179178 |
| functional organization of the rpb5 subunit shared by the three yeast rna polymerases. | rpb5, a subunit shared by the three yeast rna polymerases, combines a eukaryotic n-terminal module with a globular c-end conserved in all non-bacterial enzymes. conditional and lethal mutants of the moderately conserved eukaryotic module showed that its large n-terminal helix and a short motif at the end of the module are critical in vivo. lethal or conditional mutants of the c-terminal globe altered the binding of rpb5 to rpb1-beta25/26 (prolonging the bridge helix) and rpb1-alpha44/47 (ahead o ... | 2007 | 17179178 |
| crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein c from thermus thermophilus. | the gram-negative aerobic eubacterium thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in japan. the molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. the molybdenum-cofactor biosynthesis protein c derived from t. thermophilus was crystallized in two different space grou ... | 2007 | 17183168 |
| crystallization and preliminary x-ray diffraction analysis of an escherichia coli trna(gly) acceptor-stem microhelix. | the trna(gly) and glycyl-trna synthetase (glyrs) system is an evolutionary special case within the class ii aminoacyl-trna synthetases because two divergent types of glyrs exist: an archaebacterial/human type and an eubacterial type. the trna identity elements which determine the correct aminoacylation process are located in the aminoacyl domain of trna(gly). to obtain further insight concerning structural investigation of the identity elements, the escherichia coli seven-base-pair trna(gly) acc ... | 2007 | 17183173 |
| crystallization and preliminary x-ray diffraction analysis of an escherichia coli trna(gly) acceptor-stem microhelix. | the trna(gly) and glycyl-trna synthetase (glyrs) system is an evolutionary special case within the class ii aminoacyl-trna synthetases because two divergent types of glyrs exist: an archaebacterial/human type and an eubacterial type. the trna identity elements which determine the correct aminoacylation process are located in the aminoacyl domain of trna(gly). to obtain further insight concerning structural investigation of the identity elements, the escherichia coli seven-base-pair trna(gly) acc ... | 2007 | 17183173 |
| catalases are nad(p)h-dependent tellurite reductases. | reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. the well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. many catalases also bind the cofactors nadph and nadh tenaciously, but, surprising ... | 2006 | 17183702 |
| experimental rugged fitness landscape in protein sequence space. | the fitness landscape in sequence space determines the process of biomolecular evolution. to plot the fitness landscape of protein function, we carried out in vitro molecular evolution beginning with a defective fd phage carrying a random polypeptide of 139 amino acids in place of the g3p minor coat protein d2 domain, which is essential for phage infection. after 20 cycles of random substitution at sites 12-130 of the initial random polypeptide and selection for infectivity, the selected phage s ... | 2006 | 17183728 |
| temporal regulation of viral transcription during development of thermus thermophilus bacteriophage phiys40. | regulation of gene expression of lytic bacteriophage varphiys40 that infects the thermophilic bacterium thermus thermophilus was investigated and three temporal classes of phage genes, early, middle, and late, were revealed. varphiys40 does not encode a (rnap) and must rely on host rnap for transcription of its genes. bioinformatic analysis using a model of thermus promoters predicted 43 putative sigma(a)-dependent -10/-35 class phage promoters. a randomly chosen subset of those promoters was sh ... | 2007 | 17187825 |
| high performance computing in biology: multimillion atom simulations of nanoscale systems. | computational methods have been used in biology for sequence analysis (bioinformatics), all-atom simulation (molecular dynamics and quantum calculations), and more recently for modeling biological networks (systems biology). of these three techniques, all-atom simulation is currently the most computationally demanding, in terms of compute load, communication speed, and memory load. breakthroughs in electrostatic force calculation and dynamic load balancing have enabled molecular dynamics simulat ... | 2007 | 17187988 |
| high performance computing in biology: multimillion atom simulations of nanoscale systems. | computational methods have been used in biology for sequence analysis (bioinformatics), all-atom simulation (molecular dynamics and quantum calculations), and more recently for modeling biological networks (systems biology). of these three techniques, all-atom simulation is currently the most computationally demanding, in terms of compute load, communication speed, and memory load. breakthroughs in electrostatic force calculation and dynamic load balancing have enabled molecular dynamics simulat ... | 2007 | 17187988 |
| glucosylglycerate biosynthesis in the deepest lineage of the bacteria: characterization of the thermophilic proteins gpgs and gpgp from persephonella marina. | the pathway for the synthesis of glucosylglycerate (gg) in the thermophilic bacterium persephonella marina is proposed based on the activities of recombinant glucosyl-3-phosphoglycerate (gpg) synthase (gpgs) and glucosyl-3-phosphoglycerate phosphatase (gpgp). the sequences of gpgs and gpgp from the cold-adapted bacterium methanococcoides burtonii were used to identify the homologues in the genome of p. marina, which were separately cloned and overexpressed as his-tagged proteins in escherichia c ... | 2007 | 17189358 |
| glucosylglycerate biosynthesis in the deepest lineage of the bacteria: characterization of the thermophilic proteins gpgs and gpgp from persephonella marina. | the pathway for the synthesis of glucosylglycerate (gg) in the thermophilic bacterium persephonella marina is proposed based on the activities of recombinant glucosyl-3-phosphoglycerate (gpg) synthase (gpgs) and glucosyl-3-phosphoglycerate phosphatase (gpgp). the sequences of gpgs and gpgp from the cold-adapted bacterium methanococcoides burtonii were used to identify the homologues in the genome of p. marina, which were separately cloned and overexpressed as his-tagged proteins in escherichia c ... | 2007 | 17189358 |
| mass spectrometry-based detection of transfer rnas by their signature endonuclease digestion products. | the separation of biologically active, pure, and specific trnas is difficult due to the overall similarity in secondary and tertiary structures of different trnas. because prior methods do not facilitate high-resolution separations of the extremely complex mixture represented by a cellular trna population, global studies of trna identity and/or abundance are difficult. we have discovered that the enzymatic digestion of an individual trna by a ribonuclease (e.g., rnase t1) will generate digestion ... | 2007 | 17194720 |
| escherichia coli as a platform for functional expression of plant p450 carotene hydroxylases. | carotenoids and their derivatives are essential for growth, development, and signaling in plants and have an added benefit as nutraceuticals in food crops. despite the importance of the biosynthetic pathway, there remain open questions regarding some of the later enzymes in the pathway. the cyp97 family of p450 enzymes was predicted to function in carotene ring hydroxylation, to convert provitamin a carotenes to non-provitamin a xanthophylls. however, substrate specificity was difficult to inves ... | 2007 | 17196929 |
| escherichia coli as a platform for functional expression of plant p450 carotene hydroxylases. | carotenoids and their derivatives are essential for growth, development, and signaling in plants and have an added benefit as nutraceuticals in food crops. despite the importance of the biosynthetic pathway, there remain open questions regarding some of the later enzymes in the pathway. the cyp97 family of p450 enzymes was predicted to function in carotene ring hydroxylation, to convert provitamin a carotenes to non-provitamin a xanthophylls. however, substrate specificity was difficult to inves ... | 2007 | 17196929 |
| importance of the 5 s rrna-binding ribosomal proteins for cell viability and translation in escherichia coli. | a specific complex of 5 s rrna and several ribosomal proteins is an integral part of ribosomes in all living organisms. here we studied the importance of escherichia coli genes rple, rplr and rply, encoding 5 s rrna-binding ribosomal proteins l5, l18 and l25, respectively, for cell growth, viability and translation. using recombineering to create gene replacements in the e. coli chromosome, it was shown that rple and rplr are essential for cell viability, whereas cells deleted for rply are viabl ... | 2007 | 17198710 |
| importance of the 5 s rrna-binding ribosomal proteins for cell viability and translation in escherichia coli. | a specific complex of 5 s rrna and several ribosomal proteins is an integral part of ribosomes in all living organisms. here we studied the importance of escherichia coli genes rple, rplr and rply, encoding 5 s rrna-binding ribosomal proteins l5, l18 and l25, respectively, for cell growth, viability and translation. using recombineering to create gene replacements in the e. coli chromosome, it was shown that rple and rplr are essential for cell viability, whereas cells deleted for rply are viabl ... | 2007 | 17198710 |
| structure of glnk1 with bound effectors indicates regulatory mechanism for ammonia uptake. | a binary complex of the ammonia channel amt1 from methanococcus jannaschii and its cognate p(ii) signalling protein glnk1 has been produced and characterized. complex formation is prevented specifically by the effector molecules mg-atp and 2-ketoglutarate. single-particle electron microscopy of the complex shows that glnk1 binds on the cytoplasmic side of amt1. three high-resolution x-ray structures of glnk1 indicate that the functionally important t-loop has an extended, flexible conformation i ... | 2007 | 17203075 |
| a top-down/bottom-up study of the ribosomal proteins of caulobacter crescentus. | ribosomes from the gram-negative alpha-proteobacterium caulobacter crescentus were isolated using standard methods. proteins were separated using a two-dimensional liquid chromatographic system that allowed the analysis of whole proteins by direct coupling to an esi-qtof mass spectrometer and of proteolytic digests by a number of mass spectrometric methods. the masses of 53 of 54 ribosomal proteins were directly measured. protein identifications and proposed post-translational modifications were ... | 2007 | 17203977 |
| deinococcus radiodurans rna ligase exemplifies a novel ligase clade with a distinctive n-terminal module that is important for 5'-po4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick. | deinococcus radiodurans rna ligase (drarnl) is a template-directed ligase that seals nicked duplexes in which the 3'-oh strand is rna. drarnl is a 342 amino acid polypeptide composed of a c-terminal adenylyltransferase domain fused to a distinctive 126 amino acid n-terminal module (a putative ob-fold). an alanine scan of the c domain identified 9 amino acids essential for nick ligation, which are located within nucleotidyltransferase motifs i, ia, iii, iiia, iv and v. seven mutants were dysfunct ... | 2007 | 17204483 |
| effects of dksa, grea, and greb on transcription initiation: insights into the mechanisms of factors that bind in the secondary channel of rna polymerase. | escherichia coli dksa, grea, and greb have similar structures and bind to the same location on rna polymerase (rnap), the secondary channel. we show that greb can fulfil some roles of dksa in vitro, including shifting the promoter-open complex equilibrium in the dissociation direction, thus allowing rrna promoters to respond to changes in the concentration of ppgpp and ntps. however, unlike deletion of the dksa gene, deletion of greb had no effect on rrna promoters in vivo. we show that the appa ... | 2007 | 17207814 |
| effects of dksa, grea, and greb on transcription initiation: insights into the mechanisms of factors that bind in the secondary channel of rna polymerase. | escherichia coli dksa, grea, and greb have similar structures and bind to the same location on rna polymerase (rnap), the secondary channel. we show that greb can fulfil some roles of dksa in vitro, including shifting the promoter-open complex equilibrium in the dissociation direction, thus allowing rrna promoters to respond to changes in the concentration of ppgpp and ntps. however, unlike deletion of the dksa gene, deletion of greb had no effect on rrna promoters in vivo. we show that the appa ... | 2007 | 17207814 |
| co-evolution of the archaeal trna-dependent amidotransferase gatcab with trna(asn). | the important identity elements in trna(gln) and trna(asn) for bacterial gatcab and in trna(gln) for archaeal gatde are the d-loop and the first base pair of the acceptor stem. here we show that methanothermobacter thermautotrophicus gatcab, the archaeal enzyme, is different as it discriminates asp-trna(asp) and asp-trna(asn) by use of u49, the d-loop and to a lesser extent the variable loop. since archaea possess the trna(gln)-specific amidotransferase gatde, the archaeal gatcab enzyme evolved ... | 2007 | 17214986 |
| nuclease activity of the muts homologue muts2 from thermus thermophilus is confined to the smr domain. | muts homologues are highly conserved enzymes engaged in dna mismatch repair (mmr), meiotic recombination and other dna modifications. genome sequencing projects have revealed that bacteria and plants possess a muts homologue, muts2. muts2 lacks the mismatch-recognition domain of muts, but contains an extra c-terminal region called the small muts-related (smr) domain. sequences homologous to the smr domain are annotated as 'proteins of unknown function' in various organisms ranging from bacteria ... | 2007 | 17215294 |
| recognition of ribosomal protein l11 by the protein trimethyltransferase prma. | bacterial ribosomal protein l11 is post-translationally trimethylated at multiple residues by a single methyltransferase, prma. here, we describe four structures of prma from the extreme thermophile thermus thermophilus. two apo-prma structures at 1.59 and 2.3 a resolution and a third with bound cofactor s-adenosyl-l-methionine at 1.75 a each exhibit distinct relative positions of the substrate recognition and catalytic domains, revealing how prma can position the l11 substrate for multiple, con ... | 2007 | 17215866 |
| the crystal structure of the escherichia coli amtb-glnk complex reveals how glnk regulates the ammonia channel. | amt proteins are ubiquitous channels for the conduction of ammonia in archaea, eubacteria, fungi, and plants. in escherichia coli, previous studies have indicated that binding of the pii signal transduction protein glnk to the ammonia channel amtb regulates the channel thereby controlling ammonium influx in response to the intracellular nitrogen status. here, we describe the crystal structure of the complex between amtb and glnk at a resolution of 2.5 a. this structure of pii in a complex with o ... | 2007 | 17220269 |
| atomic resolution structures of rieske iron-sulfur protein: role of hydrogen bonds in tuning the redox potential of iron-sulfur clusters. | the rieske [2fe-2s] iron-sulfur protein of cytochrome bc(1) functions as the initial electron acceptor in the rate-limiting step of the catalytic reaction. prior studies have established roles for a number of conserved residues that hydrogen bond to ligands of the [2fe-2s] cluster. we have constructed site-specific variants at two of these residues, measured their thermodynamic and functional properties, and determined atomic resolution x-ray crystal structures for the native protein at 1.2 a re ... | 2007 | 17223530 |
| complete kinetic mechanism of homoisocitrate dehydrogenase from saccharomyces cerevisiae. | the kinetic mechanism of homoisocitrate dehydrogenase from saccharomyces cerevisiae was determined using initial velocity studies in the absence and presence of product and dead end inhibitors in both reaction directions. data suggest a steady state random kinetic mechanism. the dissociation constant of the mg-homoisocitrate complex (mghic) was estimated to be 11 +/- 2 mm as measured using mg2+ as a shift reagent. initial velocity data indicate the mghic complex is the reactant in the direction ... | 2007 | 17223711 |
| visualizing the atpase cycle in a protein disaggregating machine: structural basis for substrate binding by clpb. | clpb is a ring-shaped molecular chaperone that has the remarkable ability to disaggregate stress-damaged proteins. here we present the electron cryomicroscopy reconstruction of an atp-activated clpb trap mutant, along with reconstructions of clpb in the amppnp, adp, and in the nucleotide-free state. we show that motif 2 of the clpb m domain is positioned between the d1-large domains of neighboring subunits and could facilitate a concerted, atp-driven conformational change in the aaa-1 ring. we f ... | 2007 | 17244533 |
| post-transcriptional modifications in the small subunit ribosomal rna from thermotoga maritima, including presence of a novel modified cytidine. | post-transcriptional modifications of rna are nearly ubiquitous in the principal rnas involved in translation. however, in the case of rrna the functional roles of modification are far less established than for trna, and are subject to less knowledge in terms of specific nucleoside identities and their sequence locations. post-transcriptional modifications have been studied in the ssu rrna from thermotoga maritima (optimal growth 80 degrees c), one of the most deeply branched organisms in the eu ... | 2007 | 17255199 |
| structural conservation of recf and rad50: implications for dna recognition and recf function. | recf, together with reco and recr, belongs to a ubiquitous group of recombination mediators (rms) that includes eukaryotic proteins such as rad52 and brca2. rms help maintain genome stability in the presence of dna damage by loading reca-like recombinases and displacing single-stranded dna-binding proteins. here, we present the crystal structure of recf from deinococcus radiodurans. recf exhibits a high degree of structural similarity with the head domain of rad50, but lacks its long coiled-coil ... | 2007 | 17255941 |
| asymmetric deceleration of clpb or hsp104 atpase activity unleashes protein-remodeling activity. | two members of the aaa+ superfamily, clpb and hsp104, collaborate with hsp70 and hsp40 to rescue aggregated proteins. however, the mechanisms that elicit and underlie their protein-remodeling activities remain unclear. we report that for both hsp104 and clpb, mixtures of atp and atp-gammas unexpectedly unleash activation, disaggregation and unfolding activities independent of cochaperones. mutations reveal how remodeling activities are elicited by impaired hydrolysis at individual nucleotide-bin ... | 2007 | 17259993 |
| the tail of the parg dna segregation protein remodels parf polymers and enhances atp hydrolysis via an arginine finger-like motif. | the parf protein of plasmid tp228 belongs to the ubiquitous superfamily of para atpases that drive dna segregation in bacteria. atp-bound parf polymerizes into multistranded filaments. the partner protein parg is dimeric, consisting of c-termini that interweave into a ribbon-helix-helix domain contacting the centromeric dna and unstructured n-termini. parg stimulates atp hydrolysis by parf approximately 30-fold. here, we establish that the mobile tails of parg are crucial for this enhancement an ... | 2007 | 17261809 |
| region 1.2 of the rna polymerase sigma subunit controls recognition of the -10 promoter element. | recognition of the -10 promoter consensus element by region 2 of the bacterial rna polymerase sigma subunit is a key step in transcription initiation. sigma also functions as an elongation factor, inducing transcription pausing by interacting with transcribed dna non-template strand sequences that are similar to the -10 element sequence. here, we show that the region 1.2 of escherichia coli sigma70, whose function was heretofore unknown, is strictly required for efficient recognition of the non- ... | 2007 | 17268549 |
| release factors 2 from escherichia coli and thermus thermophilus: structural, spectroscopic and microcalorimetric studies. | prokaryotic class i release factors (rfs) respond to mrna stop codons and terminate protein synthesis. they interact with the ribosomal decoding site and the peptidyl-transferase centre bridging these 75 a distant ribosomal centres. for this an elongated rf conformation, with partially unfolded core domains ii.iii.iv is required, which contrasts the known compact rf crystal structures. the crystal structure of thermus thermophilus rf2 was determined and compared with solution structure of t. the ... | 2007 | 17272297 |
| characterization of a pseudomonad 2-nitrobenzoate nitroreductase and its catabolic pathway-associated 2-hydroxylaminobenzoate mutase and a chemoreceptor involved in 2-nitrobenzoate chemotaxis. | pseudomonas fluorescens strain ku-7 is a prototype microorganism that metabolizes 2-nitrobenzoate (2-nba) via the formation of 3-hydroxyanthranilate (3-haa), a known antioxidant and reductant. the initial two steps leading to the sequential formation of 2-hydroxy/aminobenzoate and 3-haa are catalyzed by a nadph-dependent 2-nba nitroreductase (nbaa) and 2-hydroxylaminobenzoate mutase (nbab), respectively. the 216-amino-acid protein nbaa is 78% identical to a plasmid-encoded hypothetical conserved ... | 2007 | 17277060 |
| purification, crystallization and preliminary x-ray analysis of the regulatory subunit of aspartate kinase from thermus thermophilus. | aspartate kinase (ak) from thermus thermophilus, which catalyzes the first step of threonine and methionine biosynthesis, is regulated via feedback inhibition by the end product threonine. to elucidate the mechanism of regulation of ak, the regulatory subunit (the beta subunit of t. thermophilus ak) was crystallized in the presence of the inhibitor threonine. diffraction data were collected to 2.15 a at a synchrotron source. the crystal belongs to the cubic space group p4(3)32 or p4(1)32, with u ... | 2007 | 17277448 |
| comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant saccharomyces cerevisiae. | two heterologous pathways have been used to construct recombinant xylose-fermenting saccharomyces cerevisiae strains: i) the xylose reductase (xr) and xylitol dehydrogenase (xdh) pathway and ii) the xylose isomerase (xi) pathway. in the present study, the pichia stipitis xr-xdh pathway and the piromyces xi pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpresse ... | 2007 | 17280608 |
| g-ribo: a new structural motif in ribosomal rna. | analysis of the available crystal structures of the ribosome and of its subunits has revealed a new rna motif that we call g-ribo. the motif consists of two double helices positioned side-by-side and connected by an unpaired region. the juxtaposition of the two helices is kept by a complex system of tertiary interactions spread over several layers of stacked nucleotides. in the center of this arrangement, the ribose of a nucleotide from one helix is specifically packed with the ribose and the mi ... | 2007 | 17283211 |
| new bioinformatic tools for analysis of nucleotide modifications in eukaryotic rrna. | this report presents a valuable new bioinformatics package for research on rrna nucleotide modifications in the ribosome, especially those created by small nucleolar rna:protein complexes (snornps). the interactive service, which is not available elsewhere, enables a user to visualize the positions of pseudouridines, 2'-o-methylations, and base methylations in three-dimensional space in the ribosome and also in linear and secondary structure formats of ribosomal rna. our tools provide additional ... | 2007 | 17283215 |
| deinococcus glutaminyl-trna synthetase is a chimer between proteins from an ancient and the modern pathways of aminoacyl-trna formation. | glutaminyl-trna synthetase from deinococcus radiodurans possesses a c-terminal extension of 215 residues appending the anticodon-binding domain. this domain constitutes a paralog of the yqey protein present in various organisms and part of it is present in the c-terminal end of the gatb subunit of gatcab, a partner of the indirect pathway of gln-trna(gln) formation. to analyze the peculiarities of the structure-function relationship of this glnrs related to the yqey domain, a structure of the pr ... | 2007 | 17284460 |
| the structure of free l11 and functional dynamics of l11 in free, l11-rrna(58 nt) binary and l11-rrna(58 nt)-thiostrepton ternary complexes. | the l11 binding site is one of the most important functional sites in the ribosome. the n-terminal domain of l11 has been implicated as a "reversible switch" in facilitating the coordinated movements associated with ef-g-driven gtp hydrolysis. the reversible switch mechanism has been hypothesized to require conformational flexibility involving re-orientation and re-positioning of the two l11 domains, and warrants a close examination of the structure and dynamics of l11. here we report the soluti ... | 2007 | 17292917 |
| widespread occurrence and genomic context of unusually small polyketide synthase genes in microbial consortia associated with marine sponges. | numerous marine sponges harbor enormous amounts of as-yet-uncultivated bacteria in their tissues. there is increasing evidence that these symbionts play an important role in the synthesis of protective metabolites, many of which are of great pharmacological interest. in this study, genes for the biosynthesis of polyketides, one of the most important classes of bioactive natural products, were systematically investigated in 20 demosponge species from different oceans. unexpectedly, the sponge met ... | 2007 | 17293531 |
| in vitro trans-translation of thermus thermophilus: ribosomal protein s1 is not required for the early stage of trans-translation. | transfer-messenger rna (tmrna) plays a dual role as a trna and an mrna in trans-translation, during which the ribosome replaces mrna with tmrna encoding the tag-peptide. these processes have been suggested to involve several tmrna-binding proteins, including smpb and ribosomal protein s1. to investigate the molecular mechanism of trans-translation, we developed in vitro systems using purified ribosome, elongation factors, tmrna and smpb from thermus thermophilus. a stalled ribosome in complex wi ... | 2007 | 17299130 |
| bacterial toxicity of potassium tellurite: unveiling an ancient enigma. | biochemical, genetic, enzymatic and molecular approaches were used to demonstrate, for the first time, that tellurite (teo(3) (2-)) toxicity in e. coli involves superoxide formation. this radical is derived, at least in part, from enzymatic teo(3) (2-) reduction. this conclusion is supported by the following observations made in k(2)teo(3)-treated e. coli bw25113: i) induction of the ibpa gene encoding for the small heat shock protein ibpa, which has been associated with resistance to superoxide ... | 2007 | 17299591 |
| toward understanding phosphoseryl-trnacys formation: the crystal structure of methanococcus maripaludis phosphoseryl-trna synthetase. | a number of archaeal organisms generate cys-trna(cys) in a two-step pathway, first charging phosphoserine (sep) onto trna(cys) and subsequently converting it to cys-trna(cys). we have determined, at 3.2-a resolution, the structure of the methanococcus maripaludis phosphoseryl-trna synthetase (seprs), which catalyzes the first step of this pathway. the structure shows that seprs is a class ii, alpha(4) synthetase whose quaternary structure arrangement of subunits closely resembles that of the het ... | 2007 | 17301225 |
| genetic engineering of zymobacter palmae for production of ethanol from xylose. | its metabolic characteristics suggest that zymobacter palmae gen. nov., sp. nov. could serve as a useful new ethanol-fermenting bacterium, but its biotechnological exploitation will require certain genetic modifications. we therefore engineered z. palmae so as to broaden the range of its fermentable sugar substrates to include the pentose sugar xylose. the escherichia coli genes encoding the xylose catabolic enzymes xylose isomerase, xylulokinase, transaldolase, and transketolase were introduced ... | 2007 | 17308178 |
| kinetic discrimination of trna identity by the conserved motif 2 loop of a class ii aminoacyl-trna synthetase. | the selection of trnas by their cognate aminoacyl-trna synthetases is critical for ensuring the fidelity of protein synthesis. while nucleotides that comprise trna identity sets have been readily identified, their specific role in the elementary steps of aminoacylation is poorly understood. by use of a rapid kinetics analysis employing mutants in trna(his) and its cognate aminoacyl-trna synthetase, the role of trna identity in aminoacylation was investigated. while mutations in the trna anticodo ... | 2007 | 17317626 |
| the structures of antibiotics bound to the e site region of the 50 s ribosomal subunit of haloarcula marismortui: 13-deoxytedanolide and girodazole. | crystal structures of the 50 s ribosomal subunit from haloarcula marismortui complexed with two antibiotics have identified new sites at which antibiotics interact with the ribosome and inhibit protein synthesis. 13-deoxytedanolide binds to the e site of the 50 s subunit at the same location as the cca of trna, and thus appears to inhibit protein synthesis by competing with deacylated trnas for e site binding. girodazole binds near the e site region, but is somewhat buried and may inhibit trna b ... | 2007 | 17321546 |
| structure of the type iii pantothenate kinase from bacillus anthracis at 2.0 a resolution: implications for coenzyme a-dependent redox biology. | coenzyme a (coash) is the major low-molecular weight thiol in staphylococcus aureus and a number of other bacteria; the crystal structure of the s. aureus coenzyme a-disulfide reductase (coadr), which maintains the reduced intracellular state of coash, has recently been reported [mallett, t.c., wallen, j.r., karplus, p.a., sakai, h., tsukihara, t., and claiborne, a. (2006) biochemistry 45, 11278-89]. in this report we demonstrate that coash is the major thiol in bacillus anthracis; a bioinformat ... | 2007 | 17323930 |
| a retro-evolution study of cdp-6-deoxy-d-glycero-l-threo-4-hexulose-3-dehydrase (e1) from yersinia pseudotuberculosis: implications for c-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses. | cdp-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase (e1), which catalyzes c-3 deoxygenation of cdp-4-keto-6-deoxyglucose in the biosynthesis of 3,6-dideoxyhexoses, shares a modest sequence identity with other b6-dependent enzymes, albeit with two important distinctions. it is a rare example of a b6-dependent enzyme that harbors a [2fe-2s] cluster, and a highly conserved lysine that serves as an anchor for plp in most b6-dependent enzymes is replaced by histidine at position 220 in e1. since alt ... | 2007 | 17323931 |
| x-ray crystallographic analysis of the sulfur carrier protein soxy from chlorobium limicola f. thiosulfatophilum reveals a tetrameric structure. | dissimilatory oxidation of thiosulfate in the green sulfur bacterium chlorobium limicola f. thiosulfatophilum is carried out by the ubiquitous sulfur-oxidizing (sox) multi-enzyme system. in this system, soxy plays a key role, functioning as the sulfur substrate-binding protein that offers its sulfur substrate, which is covalently bound to a conserved c-terminal cysteine, to another oxidizing sox enzyme. here, we report the crystal structures of a stand-alone soxy protein of c. limicola f. thiosu ... | 2007 | 17327392 |
| directed mutagenesis identifies amino acid residues involved in elongation factor tu binding to yeast phe-trnaphe. | the co-crystal structure of thermus aquaticus elongation factor tu.guanosine 5'- [beta,gamma-imido]triphosphate (ef-tu.gdpnp) bound to yeast phe-trna(phe) reveals that ef-tu interacts with the trna body primarily through contacts with the phosphodiester backbone. twenty amino acids in the trna binding cleft of thermus thermophilus ef-tu were each mutated to structurally conservative alternatives and the affinities of the mutant proteins to yeast phe-trna(phe) determined. eleven of the 20 mutatio ... | 2007 | 17328911 |
| structure of a upf0150-family protein from thermus thermophilus hb8. | ttha0281 is a hypothetical protein from thermus thermophilus hb8 that belongs to an uncharacterized protein family, upf0150, in the pfam database and to cog1598 in the national center for biotechnology information database of clusters of orthologous groups. the x-ray crystal structure of the protein was determined by a multiple-wavelength anomalous dispersion technique and was refined at 1.9 a resolution to a final r factor of 18.5%. the ttha0281 monomer adopts an alpha-beta-beta-beta-alpha fold ... | 2007 | 17329807 |
| cloning, expression, purification, crystallization and preliminary x-ray crystallographic analysis of initiation factor 1 from mycobacterium tuberculosis. | initiation factor 1 (if-1; rv3462c) from mycobacterium tuberculosis, a component of the 30s initiation complex, was cloned and heterologously expressed in escherichia coli. the protein was purified by affinity and size-exclusion chromatography and crystallized. a complete data set has been collected to high resolution. the crystals belonged to space group p2(1)2(1)2, with two molecules per asymmetric unit which are related by translational symmetry. | 2007 | 17329809 |
| an improved definition of the rna-binding specificity of secis-binding protein 2, an essential component of the selenocysteine incorporation machinery. | by binding to secis elements located in the 3'-utr of selenoprotein mrnas, the protein sbp2 plays a key role in the assembly of the selenocysteine incorporation machinery. sbp2 contains an l7ae/l30 rna-binding domain similar to that of protein 15.5k/snu13p, which binds k-turn motifs with a 3-nt bulge loop closed by a tandem of g.a and a.g pairs. here, by selex experiments, we demonstrate the capacity of sbp2 to bind such k-turn motifs with a protruding u residue. however, we show that conversion ... | 2007 | 17332014 |
| a sigma-core interaction of the rna polymerase holoenzyme that enhances promoter escape. | the sigma subunit of bacterial rna polymerase (rnap) is required for promoter-specific transcription initiation and can also participate in downstream events. several functionally important intersubunit interactions between escherichia coli sigma(70) and the core enzyme (alpha(2)betabeta'omega) have been defined. these include an interaction between conserved region 2 of sigma(70) (sigma(2)) and the coiled-coil domain of beta' (beta' coiled-coil) that is required for sequence-specific interactio ... | 2007 | 17332752 |
| elongation factor tu3 (ef-tu3) from the kirromycin producer streptomyces ramocissimus is resistant to three classes of ef-tu-specific inhibitors. | the antibiotic kirromycin inhibits prokaryotic protein synthesis by immobilizing elongation factor tu (ef-tu) on the elongating ribosome. streptomyces ramocissimus, the producer of kirromycin, contains three tuf genes. while tuf1 and tuf2 encode kirromycin-sensitive ef-tu species, the function of tuf3 is unknown. here we demonstrate that ef-tu3, in contrast to ef-tu1 and ef-tu2, is resistant to three classes of ef-tu-targeted antibiotics: kirromycin, pulvomycin, and ge2270a. a mixture of ef-tu1 ... | 2007 | 17337575 |
| in vivo modulation of a dnaj homolog, cbpa, by cbpm. | cbpa, an escherichia coli dnaj homolog, can function as a cochaperone for the dnak/hsp70 chaperone system, and its in vitro activity can be modulated by cbpm. we discovered that cbpm specifically inhibits the in vivo activity of cbpa, preventing it from functioning in cell growth and division. furthermore, we have shown that cbpm interacts with cbpa in vivo during stationary phase, suggesting that the inhibition of activity is a result of the interaction. these results reveal that the activity o ... | 2007 | 17337578 |
| structure and kinetics of monofunctional proline dehydrogenase from thermus thermophilus. | proline dehydrogenase (prodh) and delta(1)-pyrroline-5-carboxylate dehydrogenase (p5cdh) catalyze the two-step oxidation of proline to glutamate. they are distinct monofunctional enzymes in all eukaryotes and some bacteria but are fused into bifunctional enzymes known as proline utilization a (puta) in other bacteria. here we report the first structure and biochemical data for a monofunctional prodh. the 2.0-a resolution structure of thermus thermophilus prodh reveals a distorted (betaalpha)(8) ... | 2007 | 17344208 |
| surprising arginine biosynthesis: a reappraisal of the enzymology and evolution of the pathway in microorganisms. | major aspects of the pathway of de novo arginine biosynthesis via acetylated intermediates in microorganisms must be revised in light of recent enzymatic and genomic investigations. the enzyme n-acetylglutamate synthase (nags), which used to be considered responsible for the first committed step of the pathway, is present in a limited number of bacterial phyla only and is absent from archaea. in many bacteria, shorter proteins related to the gcn5-related n-acetyltransferase family appear to acet ... | 2007 | 17347518 |
| widespread distribution of archaeal reverse gyrase in thermophilic bacteria suggests a complex history of vertical inheritance and lateral gene transfers. | reverse gyrase, an enzyme of uncertain funtion, is present in all hyperthermophilic archaea and bacteria. previous phylogenetic studies have suggested that the gene for reverse gyrase has an archaeal origin and was transferred laterally (lgt) to the ancestors of the two bacterial hyperthermophilic phyla, thermotogales and aquificales. here, we performed an in-depth analysis of the evolutionary history of reverse gyrase in light of genomic progress. we found genes coding for reverse gyrase in the ... | 2007 | 17350929 |
| degradation of alkyl methyl ketones by pseudomonas veronii mek700. | pseudomonas veronii mek700 was isolated from a biotrickling filter cleaning 2-butanone-loaded waste air. the strain is able to grow on 2-butanone and 2-hexanol. the genes for degradation of short chain alkyl methyl ketones were identified by transposon mutagenesis using a newly designed transposon, mini-tn5495, and cloned in escherichia coli. dna sequence analysis of a 15-kb fragment revealed three genes involved in methyl ketone degradation. the deduced amino acid sequence of the first gene, me ... | 2007 | 17351032 |
| in vivo and in vitro investigation of bacterial type b rnase p interaction with trna 3'-cca. | for catalysis by bacterial type b rnase p, the importance of a specific interaction with p(recursor)trna 3'-cca termini is yet unclear. we show that mutation of one of the two g residues assumed to interact with 3'-cca in type b rnase p rnas inhibits cell growth, but cell viability is at least partially restored at increased rnase p levels due to rnase p protein overexpression. the in vivo defects of the mutant enzymes correlated with an enzyme defect at low mg(2+) in vitro. for bacillus subtili ... | 2007 | 17355991 |
| genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di-myo-inositol-phosphate. | di-myo-inositol 1,1'-phosphate (dip) is a major osmoprotecting metabolite in a number of hyperthermophilic species of archaea and bacteria. although the dip biosynthesis pathway was previously proposed, genes encoding only two of the four required enzymes, inositol-1-phosphate synthase and inositol monophosphatase, were identified. in this study we used a comparative genomic analysis to predict two additional genes of this pathway (termed dipa and dipb) that remained missing. in thermotoga marit ... | 2007 | 17360515 |
| regulation of the stringent response is the essential function of the conserved bacterial g protein cgta in vibrio cholerae. | the gene encoding the conserved bacterial g protein cgta (obg) is essential for viability in every organism in which it has been studied. cgta has been reported to be involved in several diverse bacterial functions, including ribosome assembly, dna repair, sporulation, and morphological development. however, none of these functions have been identified as essential. here we show that depletion of cgta in vibrio cholerae causes global changes in gene expression that are consistent with induction ... | 2007 | 17360576 |
| transcription activation mediated by a cyclic amp receptor protein from thermus thermophilus hb8. | the extremely thermophilic bacterium thermus thermophilus hb8, which belongs to the phylum deinococcus-thermus, has an open reading frame encoding a protein belonging to the cyclic amp (camp) receptor protein (crp) family present in many bacteria. the protein named t. thermophilus crp is highly homologous to the crp family proteins from the phyla firmicutes, actinobacteria, and cyanobacteria, and it forms a homodimer and interacts with camp. crp mrna and intracellular camp were detected in this ... | 2007 | 17369302 |
| blub cannibalizes flavin to form the lower ligand of vitamin b12. | vitamin b12 (cobalamin) is among the largest known non-polymeric natural products, and the only vitamin synthesized exclusively by microorganisms. the biosynthesis of the lower ligand of vitamin b(12), 5,6-dimethylbenzimidazole (dmb), is poorly understood. recently, we discovered that a sinorhizobium meliloti gene, blub, is necessary for dmb biosynthesis. here we show that blub triggers the unprecedented fragmentation and contraction of the bound flavin mononucleotide cofactor and cleavage of th ... | 2007 | 17377583 |
| a single residue in leucyl-trna synthetase affecting amino acid specificity and trna aminoacylation. | human mitochondrial leucyl-trna synthetase (hs mt leurs) achieves high aminoacylation fidelity without a functional editing active site, representing a rare example of a class i aminoacyl-trna synthetase (aars) that does not proofread its products. previous studies demonstrated that the enzyme achieves high selectivity by using a more specific synthetic active site that is not prone to errors under physiological conditions. interestingly, the synthetic active site of hs mt leurs displays a high ... | 2007 | 17378584 |
| new photoreactive trna derivatives for probing the peptidyl transferase center of the ribosome. | three new photoreactive trna derivatives have been synthesized for use as probes of the peptidyl transferase center of the ribosome. in two of these derivatives, the 3' adenosine of yeast trna(phe) has been replaced by either 2-azidodeoxyadenosine or 2-azido-2'-o-methyl adenosine, while in a third the 3'-terminal 2-azidodeoxyadenosine of the trna is joined to puromycin via a phosphoramidate linkage to generate a photoreactive transition-state analog. all three derivatives bind to the p site of 7 ... | 2007 | 17379815 |
| ribosomal rna guanine-(n2)-methyltransferases and their targets. | five nearly universal methylated guanine-(n2) residues are present in bacterial rrna in the ribosome. to date four out of five ribosomal rna guanine-(n2)-methyltransferases are described. rsmc(yjjt) methylates g1207 of the 16s rrna. rlmg(ygjo) and rlml(ycby) are responsible for the 23s rrna m(2)g1835 and m(2)g2445 formation, correspondingly. rsmd(yhhf) is necessary for methylation of g966 residue of 16s rrna. structure of escherichia coli rsmd(yhhf) methyltransferase and the structure of the met ... | 2007 | 17389639 |
| functional anthology of intrinsic disorder. 3. ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins. | currently, the understanding of the relationships between function, amino acid sequence, and protein structure continues to represent one of the major challenges of the modern protein science. as many as 50% of eukaryotic proteins are likely to contain functionally important long disordered regions. many proteins are wholly disordered but still possess numerous biologically important functions. however, the number of experimentally confirmed disordered proteins with known biological functions is ... | 2007 | 17391016 |
| extrachromosomal element capture and the evolution of multiple replication origins in archaeal chromosomes. | in all three domains of life, dna replication begins at specialized loci termed replication origins. in bacteria, replication initiates from a single, clearly defined site. in contrast, eukaryotic organisms exploit a multitude of replication origins, dividing their genomes into an array of short contiguous units. recently, the multiple replication origin paradigm has also been demonstrated within the archaeal domain of life, with the discovery that the hyperthermophilic archaeon sulfolobus has t ... | 2007 | 17392430 |
| mechanisms for stabilisation and the maintenance of solubility in proteins from thermophiles. | the database of protein structures contains representatives from organisms with a range of growth temperatures. various properties have been studied in a search for the molecular basis of protein adaptation to higher growth temperature. charged groups have emerged as key distinguishing factors for proteins from thermophiles and mesophiles. | 2007 | 17394655 |
| discovery of a thermophilic protein complex stabilized by topologically interlinked chains. | a growing number of organisms have been discovered inhabiting extreme environments, including temperatures in excess of 100 degrees c. how cellular proteins from such organisms retain their native folds under extreme conditions is still not fully understood. recent computational and structural studies have identified disulfide bonding as an important mechanism for stabilizing intracellular proteins in certain thermophilic microbes. here, we present the first proteomic analysis of intracellular d ... | 2007 | 17395198 |
| structure probing of tmrna in distinct stages of trans-translation. | ribosomes stalled on problematic mrnas in bacterial cells can be rescued by transfer-messenger rna (tmrna), its helper protein (small protein b, smpb), and elongation factor tu (ef-tu) through a mechanism called trans-translation. in this work we used lead(ii) footprinting to probe the interactions of tmrna with smpb and other components of the translation machinery at different steps of the trans-translation cycle. ribosomes with a short nascent peptide stalled on a truncated mrna were reacted ... | 2007 | 17400816 |
| crystal structure of the map kinase binding domain and the catalytic domain of human mkp5. | map kinase phosphatases (mkps) have crucial roles in regulating the signaling activity of map kinases and are potential targets for drug discovery against human diseases. these enzymes contain a catalytic domain (cd) as well as a binding domain (bd) that help recognize the target map kinase. we report here the crystal structures at up to 2.2 a resolution of the bd and cd of human mkp5 and compare them to the known structures from other mkps. dramatic structural differences are observed between t ... | 2007 | 17400920 |
| purification, crystallization and preliminary x-ray diffraction study on pyrimidine nucleoside phosphorylase ttha1771 from thermus thermophilus hb8. | pyrimidine nucleoside phosphorylase (pynp) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide-synthesis salvage pathway. in order to study the structure-thermostability relationship of this enzyme, pynp from the extreme thermophile thermus thermophilus hb8 (ttha1771) has been cloned, overexpressed and purified. the ttha1771 protein was crystallized at 291 k using the oil-microbatch method with peg 4000 as a precipitant. a native data set was collected to 1.8 a resolution us ... | 2007 | 17401202 |
| cloning, expression, purification, crystallization and preliminary x-ray crystallographic study of molybdopterin synthase from thermus thermophilus hb8. | thermus thermophilus is a gram-negative aerobic thermophilic eubacterium which can grow at temperatures ranging from 323 to 355 k. in addition to their importance in thermostability or adaptation strategies for survival at high temperatures, the thermostable enzymes in thermophilic organisms contribute to a wide range of biotechnological applications. the molybdenum cofactor in all three kingdoms consists of a tricyclic pyranopterin termed molybdopterin that bears the cis-dithiolene group respon ... | 2007 | 17401207 |
| a unique insert of leucyl-trna synthetase is required for aminoacylation and not amino acid editing. | leucyl-trna synthetase (leurs) is a class i enzyme, which houses its aminoacylation active site in a canonical core that is defined by a rossmann nucleotide binding fold. in addition, many leurss bear a unique polypeptide insert comprised of about 50 amino acids located just upstream of the conserved kmsks sequence. the role of this leucine-specific domain (ls-domain) remains undefined. we hypothesized that this domain may be important for substrate recognition in aminoacylation and/or amino aci ... | 2007 | 17407263 |
| structures of trnas with an expanded anticodon loop in the decoding center of the 30s ribosomal subunit. | during translation, some +1 frameshift mrna sites are decoded by frameshift suppressor trnas that contain an extra base in their anticodon loops. similarly engineered trnas have been used to insert nonnatural amino acids into proteins. here, we report crystal structures of two anticodon stem-loops (asls) from trnas known to facilitate +1 frameshifting bound to the 30s ribosomal subunit with their cognate mrnas. asl(cccg) and asl(accc) (5'-3' nomenclature) form unpredicted anticodon-codon interac ... | 2007 | 17416634 |
| the active ring-like structure of seca revealed by electron crystallography: conformational change upon interaction with secb. | seca is a multifunctional protein involved in protein translocation in bacteria. the structure of seca on membrane is dramatically altered compared with that in solution, accompanying with functional changes. we previously reported the formation of a novel ring-like structure of seca on lipid layers, which may constitute part of the preprotein translocation channel. in the present work, two-dimensional crystallization of escherichia coli seca on lipid monolayers was performed to reveal the struc ... | 2007 | 17419072 |
| kinetic and spectroscopic characterization of type ii isopentenyl diphosphate isomerase from thermus thermophilus: evidence for formation of substrate-induced flavin species. | type ii isopentenyl diphosphate (ipp) isomerase catalyzes the interconversion of ipp and dimethylallyl diphosphate (dmapp). although the reactions catalyzed by the type ii enzyme and the well-studied type i ipp isomerase are identical, the type ii protein requires reduced flavin for activity. the chemical mechanism, including the role of flavin, has not been established for type ii ipp isomerase. recombinant type ii ipp isomerase from thermus thermophilus hb27 was purified by ni2+ affinity chrom ... | 2007 | 17428035 |
| mechanism of chimera formation during the multiple displacement amplification reaction. | multiple displacement amplification (mda) is a method used for amplifying limiting dna sources. the high molecular weight amplified dna is ideal for dna library construction. while this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of mda to generate chimeric dna rearrangements in the amplified dna. determining the source of the dna rearrangements would be an important step towards reducing or eliminating them. | 2007 | 17430586 |
| a tale of two oxidation states: bacterial colonization of arsenic-rich environments. | microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. finding a means of preserving natural environments-including ground and surface waters-from arsenic constitutes a major challenge facing modern society. although this metalloid is ubiquitous on earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. in-depth exploration of the genome of the beta ... | 2007 | 17432936 |
| hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker. | hsp70 chaperones assist in protein folding, disaggregation, and membrane translocation by binding to substrate proteins with an atp-regulated affinity that relies on allosteric coupling between atp-binding and substrate-binding domains. we have studied single- and two-domain versions of the e. coli hsp70, dnak, to explore the mechanism of interdomain communication. we show that the interdomain linker controls atpase activity by binding to a hydrophobic cleft between subdomains ia and iia. furthe ... | 2007 | 17434124 |
| structural basis for converting a general transcription factor into an operon-specific virulence regulator. | rfah, a paralog of the general transcription factor nusg, is recruited to elongating rna polymerase at specific regulatory sites. the x-ray structure of escherichia coli rfah reported here reveals two domains. the n-terminal domain displays high similarity to that of nusg. in contrast, the alpha-helical coiled-coil c domain, while retaining sequence similarity, is strikingly different from the beta barrel of nusg. to our knowledge, such an all-beta to all-alpha transition of the entire domain is ... | 2007 | 17434131 |