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complete genome sequence of the extremely acidophilic methanotroph isolate v4, methylacidiphilum infernorum, a representative of the bacterial phylum verrucomicrobia.the phylum verrucomicrobia is a widespread but poorly characterized bacterial clade. although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. we have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the verrucomicrobia, strain v4, isolated from the hell's gate ...200818593465
nitrogen metabolism in haloarchaea.the nitrogen cycle (n-cycle), principally supported by prokaryotes, involves different redox reactions mainly focused on assimilatory purposes or respiratory processes for energy conservation. as the n-cycle has important environmental implications, this biogeochemical cycle has become a major research topic during the last few years. however, although n-cycle metabolic pathways have been studied extensively in bacteria or eukarya, relatively little is known in the archaea. halophilic archaea ar ...200818593475
crystal structure of rimi from salmonella typhimurium lt2, the gnat responsible for n(alpha)-acetylation of ribosomal protein s18.the three ribosomal proteins l7, s5, and s18 are included in the rare subset of prokaryotic proteins that are known to be n(alpha)-acetylated. the gcn5-related n-acetyltransferase (gnat) protein rimi, responsible for the n(alpha)-acetylation of the ribosomal protein s18, was cloned from salmonella typhimurium lt2 (rimi(st)), overexpressed, and purified to homogeneity. steady-state kinetic parameters for rimi(st) were determined for accoa and a peptide substrate consisting of the first six amino ...200818596200
seca, the motor of the secretion machine, binds diverse partners on one interactive surface.in all living cells, regulated passage across membranes of specific proteins occurs through a universally conserved secretory channel. in bacteria and chloroplasts, the energy for the mechanical work of moving polypeptides through that channel is provided by seca, a regulated atpase. here, we use site-directed spin labeling and electron paramagnetic resonance spectroscopy to identify the interactive surface used by seca for each of the diverse binding partners encountered during the dynamic cycl ...200818602400
iron-sulfur cluster n5 is coordinated by an hxxxcxxcxxxxxc motif in the nuog subunit of escherichia coli nadh:quinone oxidoreductase (complex i).nadh:quinone oxidoreductase (complex i) plays a central role in cellular energy metabolism, and its dysfunction is found in numerous human mitochondrial diseases. although the understanding of its structure and function has been limited, the x-ray crystal structure of the hydrophilic part of thermus thermophilus complex i recently became available. it revealed the localization of all redox centers, including 9 iron-sulfur clusters and their coordinating ligands, and confirmed the predictions mos ...200818603533
tbmp42 is a structure-sensitive ribonuclease that likely follows a metal ion catalysis mechanism.rna editing in african trypanosomes is characterized by a uridylate-specific insertion and/or deletion reaction that generates functional mitochondrial transcripts. the process is catalyzed by a multi-enzyme complex, the editosome, which consists of approximately 20 proteins. while for some of the polypeptides a contribution to the editing reaction can be deduced from their domain structure, the involvement of other proteins remains elusive. tbmp42, is a component of the editosome that is charac ...200818603593
purification, crystallization and preliminary x-ray diffraction analysis of adenosine triphosphate sulfurylase (atps) from the sulfate-reducing bacterium desulfovibrio desulfuricans atcc 27774.native zinc/cobalt-containing atp sulfurylase (atps; ec 2.7.7.4; mgatp:sulfate adenylyltransferase) from desulfovibrio desulfuricans atcc 27774 was purified to homogeneity and crystallized. the orthorhombic crystals diffracted to beyond 2.5 a resolution and the x-ray data collected should allow the determination of the structure of the zinc-bound form of this atps. although previous biochemical studies of this protein indicated the presence of a homotrimer in solution, a dimer was found in the a ...200818607083
multiple-site trimethylation of ribosomal protein l11 by the prma methyltransferase.ribosomal protein l11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. in escherichia coli, the lysine methyltransferase prma trimethylates the n-terminal alpha-amino group and the epsilon-amino groups of lys3 and lys39. here, we report four prma-l11 complex structures in different orientations with respect to the prma active site. two structures capture the l11 n-terminal alpha-amino grou ...200818611379
complex i within oxidatively stressed bovine heart mitochondria is glutathionylated on cys-531 and cys-704 of the 75-kda subunit: potential role of cys residues in decreasing oxidative damage.complex i has reactive thiols on its surface that interact with the mitochondrial glutathione pool and are implicated in oxidative damage in many pathologies. however, the cys residues and the thiol modifications involved are not known. here we investigate complex i thiol modification within oxidatively stressed mammalian mitochondria, containing physiological levels of glutathione and glutaredoxin 2. in mitochondria incubated with the thiol oxidant diamide, complex i is only glutathionylated on ...200818611857
ribosome performance is enhanced by a rich cluster of pseudouridines in the a-site finger region of the large subunit.the large subunit rrna in eukaryotes contains an unusually dense cluster of 8-10 pseudouridine (psi) modifications located in a three-helix structure (h37-h39) implicated in several functions. this region is dominated by a long flexible helix (h38) known as the "a-site finger" (asf). the asf protrudes from the large subunit just above the a-site of trna binding, interacts with 5 s rrna and trna, and through the terminal loop, forms a bridge (b1a) with the small subunit. in yeast, the three-helix ...200818611858
different aa-trnas are selected uniformly on the ribosome.ten e. coli aminoacyl-trnas (aa-trnas) were assessed for their ability to decode cognate codons on e. coli ribosomes by using three assays that evaluate the key steps in the decoding pathway. despite a wide variety of structural features, each aa-trna exhibited similar kinetic and thermodynamic properties in each assay. this surprising kinetic and thermodynamic uniformity is likely to reflect the importance of ribosome conformational changes in defining the rates and affinities of the decoding p ...200818614050
substrate specificity and structure of human aminoadipate aminotransferase/kynurenine aminotransferase ii.kat (kynurenine aminotransferase) ii is a primary enzyme in the brain for catalysing the transamination of kynurenine to kyna (kynurenic acid). kyna is the only known endogenous antagonist of the n-methyl-d-aspartate receptor. the enzyme also catalyses the transamination of aminoadipate to alpha-oxoadipate; therefore it was initially named aadat (aminoadipate aminotransferase). as an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocyt ...200818620547
the 3d rrna modification maps database: with interactive tools for ribosome analysis.the 3d rrna modification maps database is the first general resource of information about the locations of modified nucleotides within the 3d structure of the full ribosome, with mrna and trnas in the a-, p- and e-sites. the database supports analyses for several model organisms, including higher eukaryotes, and enables users to construct 3d maps for other organisms. data are provided for human and plant (arabidopsis) ribosomes, and for other representative organisms from eubacteria, archaea and ...200817947322
the 3d rrna modification maps database: with interactive tools for ribosome analysis.the 3d rrna modification maps database is the first general resource of information about the locations of modified nucleotides within the 3d structure of the full ribosome, with mrna and trnas in the a-, p- and e-sites. the database supports analyses for several model organisms, including higher eukaryotes, and enables users to construct 3d maps for other organisms. data are provided for human and plant (arabidopsis) ribosomes, and for other representative organisms from eubacteria, archaea and ...200817947322
probing the paracoccus denitrificans cytochrome c(1)-cytochrome c(552) interaction by mutagenesis and fast kinetics.electron transfer (et) between paracoccus denitrificans cytochrome (cyt) c(1) and cytochrome c(552) was studied using the soluble redox fragments cyt c(1cf) and cyt c(552f). a new ruthenium cyt c(552f) derivative labeled at c23 (ru(z)-23-c(552f)) was designed to measure rapid electron transfer with cyt c(1cf) in the physiological direction using flash photolysis. the bimolecular rate constant k(12) decreased rapidly with ionic strength above 40 mm, consistent with a diffusional process guided by ...200819006325
a short-oligonucleotide microarray that allows improved detection of gastrointestinal tract microbial communities.the human gastrointestinal (gi) tract contains a diverse collection of bacteria, most of which are unculturable by conventional microbiological methods. increasingly molecular profiling techniques are being employed to examine this complex microbial community. the purpose of this study was to develop a microarray technique based on 16s ribosomal gene sequences for rapidly monitoring the microbial population of the gi tract.200819014434
prevalence of pfmdr1, pfcrt, pfdhfr and pfdhps mutations associated with drug resistance, in luanda, angola.malaria is the infectious disease causing the highest morbidity and mortality in angola and due to widespread chloroquine (cq) resistance, the country has recently changed its first-line treatment recommendations for uncomplicated malaria, from cq to artemisinin combination therapies (act) in adults, and sulphadoxine/pyrimethamine (s/p) in pregnant women. loss of sp sensitivity is, however, progressing rapidly in africa and, in this study, were investigated a number of molecular markers associat ...200819014684
insights into translational termination from the structure of rf2 bound to the ribosome.the termination of protein synthesis occurs through the specific recognition of a stop codon in the a site of the ribosome by a release factor (rf), which then catalyzes the hydrolysis of the nascent protein chain from the p-site transfer rna. here we present, at a resolution of 3.5 angstroms, the crystal structure of rf2 in complex with its cognate uga stop codon in the 70s ribosome. the structure provides insight into how rf2 specifically recognizes the stop codon; it also suggests a model for ...200818988853
rv0802c from mycobacterium tuberculosis: the first structure of a succinyltransferase with the gnat fold.gene rv0802c from mycobacterium tuberculosis encodes a 218-amino-acid protein and is annotated as a hypothetical protein with homology to gcn5-related n-acetyltransferases. the structure of rv0802c was determined in an unliganded form to 2.0 a resolution utilizing single-wavelength anomalous dispersion from a samarium soak that resulted in a single bound sm(3+):citrate(2) complex. the structure confirms that rv0802c exhibits the gcn5-related n-acetyltransferase fold and revealed a tetramer compo ...200818997321
core structure of the yeast spt4-spt5 complex: a conserved module for regulation of transcription elongation.the spt4-spt5 complex is an essential rna polymerase ii elongation factor found in all eukaryotes and important for gene regulation. we report here the crystal structure of saccharomyces cerevisiae spt4 bound to the ngn domain of spt5. this structure reveals that spt4-spt5 binding is governed by an acid-dipole interaction between spt5 and spt4. mutations that disrupt this interaction disrupt the complex. residues forming this pivotal interaction are conserved in the archaeal homologs of spt4 and ...200819000817
comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting saccharomyces cerevisiae strains.ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. this process would greatly benefit from recombinant saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. different pathways can be introduced in s. cerevisiae to provide arabinose and xylose utilisation. in this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation p ...200818947407
structure of the yeast vacuolar atpase.the subunit architecture of the yeast vacuolar atpase (v-atpase) was analyzed by single particle transmission electron microscopy and electrospray ionization (esi) tandem mass spectrometry. a three-dimensional model of the intact v-atpase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. images of yeast v-atpase decorated with monoclonal antibodies against subunits a, e, and g position subunit a within the pseudo-hexagonal arrangement in the v1, the ...200818955482
the p. furiosus mre11/rad50 complex promotes 5' strand resection at a dna double-strand break.the mre11/rad50 complex has been implicated in the early steps of dna double-strand break (dsb) repair through homologous recombination in several organisms. however, the enzymatic properties of this complex are incompatible with the generation of 3' single-stranded dna for recombinase loading and strand exchange. in thermophilic archaea, the mre11 and rad50 genes cluster in an operon with genes encoding a helicase, hera, and a 5' to 3' exonuclease, nura, suggesting a common function. here we sh ...200818957200
the rna polymerase "switch region" is a target for inhibitors.the alpha-pyrone antibiotic myxopyronin (myx) inhibits bacterial rna polymerase (rnap). here, through a combination of genetic, biochemical, and structural approaches, we show that myx interacts with the rnap "switch region"--the hinge that mediates opening and closing of the rnap active center cleft--to prevent interaction of rnap with promoter dna. we define the contacts between myx and rnap and the effects of myx on rnap conformation and propose that myx functions by interfering with opening ...200818957204
automated motif extraction and classification in rna tertiary structures.we used a novel graph-based approach to extract rna tertiary motifs. we cataloged them all and clustered them using an innovative graph similarity measure. we applied our method to three widely studied structures: haloarcula marismortui 50s (h.m 50s), escherichia coli 50s (e. coli 50s), and thermus thermophilus 16s (t.th 16s) rnas. we identified 10 known motifs without any prior knowledge of their shapes or positions. we additionally identified four putative new motifs.200818957493
sequence and structural evolution of the ksga/dim1 methyltransferase family.one of the 60 or so genes conserved in all domains of life is the ksga/dim1 orthologous group. enzymes from this family perform the same post-transcriptional nucleotide modification in ribosome biogenesis, irrespective of organism. despite this common function, divergence has enabled some family members to adopt new and sometimes radically different functions. for example, in s. cerevisiae dim1 performs two distinct functions in ribosome biogenesis, while human mttfb is not only an rrna methyltr ...200818959795
translation initiation factor if1 of bacillus stearothermophilus and thermus thermophilus substitute for escherichia coli if1 in vivo and in vitro without a direct if1-if2 interaction.bacterial translation initiation factor if1 is homologous to archaeal aif1a and eukaryal eif1a, which form a complex with their homologous if2-like factors (aif5b and eif5b respectively) during initiation of protein synthesis. a similar if1-if2 interaction is assumed to occur in all bacteria and supported by cross-linking data and stabilization of the 30s-if2 interaction by if1. here we compare escherichia coli if1 with thermophilic factors from bacillus stearothermophilus and thermus thermophil ...200818976282
mutations in conserved helix 69 of 23s rrna of thermus thermophilus that affect capreomycin resistance but not posttranscriptional modifications.translocation during the elongation phase of protein synthesis involves the relative movement of the 30s and 50s ribosomal subunits. this movement is the target of tuberactinomycin antibiotics. here, we describe the isolation and characterization of mutants of thermus thermophilus selected for resistance to the tuberactinomycin antibiotic capreomycin. two base substitutions, a1913u and mu1915g, and a single base deletion, deltamu1915, were identified in helix 69 of 23s rrna, a structural element ...200818805973
mechanism of 4-nitrophenol oxidation in rhodococcus sp. strain pn1: characterization of the two-component 4-nitrophenol hydroxylase and regulation of its expression.4-nitrophenol (4-np) is a toxic product of the hydrolysis of organophosphorus pesticides such as parathion in soil. rhodococcus sp. strain pn1 degrades 4-np via 4-nitrocatechol (4-nc) for use as the sole carbon, nitrogen, and energy source. a 5-kb ecori dna fragment previously cloned from pn1 contained a gene cluster (nphra1a2) involved in 4-np oxidation. from sequence analysis, this gene cluster is expected to encode an arac/xyls family regulatory protein (nphr) and a two-component 4-np hydroxy ...200818805976
structural analysis of fad synthetase from corynebacterium ammoniagenes.the prokaryotic fad synthetase family - a group of bifunctional enzymes that catalyse riboflavin phosphorylation and fmn adenylylation within a single polypeptide chain- was analysed in terms of sequence and structure.200818811972
endosymbiont gene functions impaired and rescued by polymerase infidelity at poly(a) tracts.among host-dependent bacteria that have evolved by extreme reductive genome evolution, long-term bacterial endosymbionts of insects have the smallest (160-790 kb) and most a + t-rich (>70%) bacterial genomes known to date. these genomes are riddled with poly(a) tracts, and 5-50% of genes contain tracts of 10 as or more. here, we demonstrate transcriptional slippage at poly(a) tracts within genes of buchnera aphidicola associated with aphids and blochmannia pennsylvanicus associated with ants. se ...200818815381
protein co-evolution, co-adaptation and interactions.co-evolution has an important function in the evolution of species and it is clearly manifested in certain scenarios such as host-parasite and predator-prey interactions, symbiosis and mutualism. the extrapolation of the concepts and methodologies developed for the study of species co-evolution at the molecular level has prompted the development of a variety of computational methods able to predict protein interactions through the characteristics of co-evolution. particularly successful have bee ...200818818697
yeast ribosomal protein l10 helps coordinate trna movement through the large subunit.yeast ribosomal protein l10 (e. coli l16) is located at the center of a topological nexus that connects many functional regions of the large subunit. this essential protein has previously been implicated in processes as diverse as ribosome biogenesis, translational fidelity and mrna stability. here, the inability to maintain the yeast killer virus was used as a proxy for large subunit defects to identify a series of l10 mutants. these mapped to roughly four discrete regions of the protein. a det ...200818824477
kinetic and thermodynamic studies of peptidyltransferase in ribosomes from the extreme thermophile thermus thermophilus.throughout evolution, emerging organisms survived by adapting existing biochemical processes to new reaction conditions. simple protein enzymes balanced changes in structural stability with changes that permitted optimal catalysis by adjustments in both entropic and enthalpic contributions to the free energy of activation for the reaction. study of adaptive mechanisms by large multicomponent enzymes such as the ribosome has been largely unexplored. here we have determined the kinetic and thermod ...200818824514
identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of lactobacillus brevis atcc 8287.lactobacillus brevis atcc 8287 is covered by a regular surface (s-) layer consisting of a 435 amino acid protein slpa. this protein is completely unrelated in sequence to the previously characterized s-layer proteins of lactobacillus acidophilus group.200818828902
the crystal structure of desulfovibrio vulgaris dissimilatory sulfite reductase bound to dsrc provides novel insights into the mechanism of sulfate respiration.sulfate reduction is one of the earliest types of energy metabolism used by ancestral organisms to sustain life. despite extensive studies, many questions remain about the way respiratory sulfate reduction is associated with energy conservation. a crucial enzyme in this process is the dissimilatory sulfite reductase (dsir), which contains a unique siroheme-[4fe4s] coupled cofactor. here, we report the structure of desulfoviridin from desulfovibrio vulgaris, in which the dsir dsrab (sulfite reduc ...200818829451
the mycoplasma pneumoniae mpn229 gene encodes a protein that selectively binds single-stranded dna and stimulates recombinase a-mediated dna strand exchange.mycoplasma pneumoniae has previously been characterized as a micro-organism that is genetically highly stable. in spite of this genetic stability, homologous dna recombination has been hypothesized to lie at the basis of antigenic variation of the major surface protein, p1, of m. pneumoniae. in order to identify the proteins that may be involved in homologous dna recombination in m. pneumoniae, we set out to characterize the mpn229 open reading frame (orf), which bears sequence similarity to the ...200818831760
ribosomal protein l3 functions as a 'rocker switch' to aid in coordinating of large subunit-associated functions in eukaryotes and archaea.although ribosomal rnas (rrnas) comprise the bulk of the ribosome and carry out its main functions, ribosomal proteins also appear to play important structural and functional roles. many ribosomal proteins contain long, nonglobular domains that extend deep into the rrna cores. in eukaryotes and archaea, ribosomal protein l3 contains two such extended domains tethered to a common globular hub, thus providing an excellent model to address basic questions relating to ribosomal protein structure/fun ...200818832371
structure of a sigma28-regulated nonflagellar virulence protein from campylobacter jejuni.campylobacter jejuni, a gram-negative motile bacterium, is a leading cause of human gastrointestinal infections. although the mechanism of c.jejuni-mediated enteritis appears to be multifactorial, flagella play complex roles in the virulence of this human pathogen. cj0977 is a recently identified virulence factor in c. jejuni and is expressed by a sigma(28) promoter that controls late genes in the flagellar regulon. a cj0977 mutant strain is fully motile but significantly reduced in the invasion ...200818835274
a unique combination of genetic systems for the synthesis of trehalose in rubrobacter xylanophilus: properties of a rare actinobacterial tret.trehalose is the primary organic solute in rubrobacter xylanophilus under all conditions tested, including those for optimal growth. we detected genes of four different pathways for trehalose synthesis in the genome of this organism, namely, the trehalose-6-phosphate synthase (tps)/trehalose-6-phosphate phosphatase (tpp), tres, trey/trez, and tret pathways. moreover, r. xylanophilus is the only known member of the phylum actinobacteria to harbor tret. the tps sequence is typically bacterial, but ...200818835983
crystal structure of muts2 endonuclease domain and the mechanism of homologous recombination suppression.dna recombination events need to be strictly regulated, because an increase in the recombinational frequency causes unfavorable alteration of genetic information. recent studies revealed the existence of a novel anti-recombination enzyme, muts2. however, the mechanism by which muts2 inhibits homologous recombination has been unknown. previously, we found that thermus thermophilus muts2 (ttmuts2) harbors an endonuclease activity and that this activity is confined to the c-terminal domain, whose a ...200818838375
rnomics and modomics in the halophilic archaea haloferax volcanii: identification of rna modification genes.naturally occurring rnas contain numerous enzymatically altered nucleosides. differences in rna populations (rnomics) and pattern of rna modifications (modomics) depends on the organism analyzed and are two of the criteria that distinguish the three kingdoms of life. if the genomic sequences of the rna molecules can be derived from whole genome sequence information, the modification profile cannot and requires or direct sequencing of the rnas or predictive methods base on the presence or absence ...200818844986
proton-dependent electron transfer from cua to heme a and altered epr spectra in mutants close to heme a of cytochrome oxidase.eukaryotic cytochrome c oxidase (cco) and homologous prokaryotic forms of rhodobacter and paraccocus differ in the epr spectrum of heme a. it was noted that a histidine ligand of heme a (h102) is hydrogen bonded to serine in rhodobacter (s44) and paraccocus ccos, in contrast to glycine in the bovine enzyme. mutation of s44 to glycine shifts the heme a epr signal from g(z) = 2.82 to 2.86, closer to bovine heme a at 3.03, without modifying other properties. mutation to aspartate, however, results ...200818847227
the high-resolution nmr structure of the early folding intermediate of the thermus thermophilus ribonuclease h.elucidation of the high-resolution structures of folding intermediates is a necessary but difficult step toward the ultimate understanding of the mechanism of protein folding. here, using hydrogen-exchange-directed protein engineering, we populated the folding intermediate of the thermus thermophilus ribonuclease h, which forms before the rate-limiting transition state, by removing the unfolded regions of the intermediate, including an alpha-helix and two beta-strands (51 folded residues). using ...200818848567
the bet v 1 fold: an ancient, versatile scaffold for binding of large, hydrophobic ligands.the major birch pollen allergen, bet v 1, is a member of the ubiquitous pr-10 family of plant pathogenesis-related proteins. in recent years, a number of diverse plant proteins with low sequence similarity to bet v 1 was identified. in addition, determination of the bet v 1 structure revealed the existence of a large superfamily of structurally related proteins. in this study, we aimed to identify and classify all bet v 1-related structures from the protein data bank and all bet v 1-related sequ ...200818922149
conformational transition of sec machinery inferred from bacterial secye structures.over 30% of proteins are secreted across or integrated into membranes. their newly synthesized forms contain either cleavable signal sequences or non-cleavable membrane anchor sequences, which direct them to the evolutionarily conserved sec translocon (secyeg in prokaryotes and sec61, comprising alpha-, gamma- and beta-subunits, in eukaryotes). the translocon then functions as a protein-conducting channel. these processes of protein localization occur either at or after translation. in bacteria, ...200818923527
rare codons cluster.most amino acids are encoded by more than one codon. these synonymous codons are not used with equal frequency: in every organism, some codons are used more commonly, while others are more rare. though the encoded protein sequence is identical, selective pressures favor more common codons for enhanced translation speed and fidelity. however, rare codons persist, presumably due to neutral drift. here, we determine whether other, unknown factors, beyond neutral drift, affect the selection and/or d ...200818923675
toward a chemical mechanism of proton pumping by the b-type cytochrome c oxidases: application of density functional theory to cytochrome ba3 of thermus thermophilus.a mechanism for proton pumping by the b-type cytochrome c oxidases is presented in which one proton is pumped in conjunction with the weakly exergonic, two-electron reduction of fe-bound o 2 to the fe-cu bridging peroxodianion and three protons are pumped in conjunction with the highly exergonic, two-electron reduction of fe(iii)- (-)o-o (-)-cu(ii) to form water and the active oxidized enzyme, fe(iii)- (-)oh,cu(ii). the scheme is based on the active-site structure of cytochrome ba 3 from thermus ...200818928258
purification, crystallization and preliminary x-ray diffraction analysis of the cbs-domain pair from the methanococcus jannaschii protein mj0100.cbs domains are small protein motifs consisting of a three-stranded beta-sheet and two alpha-helices that are present in proteins of all kingdoms of life and in proteins with completely different functions. several genetic diseases in humans have been associated with mutations in their sequence, which has made them promising targets for rational drug design. the c-terminal domain of the methanococcus jannaschii protein mj0100 includes a cbs-domain pair and has been overexpressed, purified and cr ...200818931440
three crystal forms of the bifunctional enzyme proline utilization a (puta) from bradyrhizobium japonicum.proline utilization a proteins (putas) are large (1000-1300 residues) membrane-associated bifunctional flavoenzymes that catalyze the two-step oxidation of proline to glutamate by the sequential action of proline dehydrogenase and delta(1)-pyrroline-5-carboxylate dehydrogenase domains. here, the first successful crystallization efforts for a puta protein are described. three crystal forms of puta from bradyrhizobium japonicum are reported: apparent tetragonal, hexagonal and centered monoclinic. ...200818931443
path of nascent polypeptide in exit tunnel revealed by molecular dynamics simulation of ribosome.molecular dynamics simulations were carried out on thermus thermophilus 70s ribosome with and without a nascent polypeptide inside the exit tunnel. modeling of the polypeptide in the tunnel revealed two possible paths: one over arg92 of l22 and one under (from the viewpoint of 50s on top of 30s). a strong interaction between l4 and arg92 was observed without the polypeptide and when it passed over arg92. however, when the polypeptide passed under, arg92 repositioned to interact with ade2059 of 2 ...200818936244
ssb as an organizer/mobilizer of genome maintenance complexes.when duplex dna is altered in almost any way (replicated, recombined, or repaired), single strands of dna are usually intermediates, and single-stranded dna binding (ssb) proteins are present. these proteins have often been described as inert, protective dna coatings. continuing research is demonstrating a far more complex role of ssb that includes the organization and/or mobilization of all aspects of dna metabolism. escherichia coli ssb is now known to interact with at least 14 other proteins ...200818937104
a full-length group 1 bacterial sigma factor adopts a compact structure incompatible with dna binding.the sigma factors are the key regulators of bacterial transcription initiation. through direct read-out of promoter dna sequence, they recruit the core rna polymerase to sites of initiation, thereby dictating the rna polymerase promoter-specificity. the group 1 sigma factors, which direct the vast majority of transcription initiation during log phase growth and are essential for viability, are autoregulated by an n-terminal sequence known as sigma1.1. we report the solution structure of thermoto ...200818940669
glycine cleavage system: reaction mechanism, physiological significance, and hyperglycinemia.the glycine cleavage system catalyzes the following reversible reaction: glycine + h(4)folate + nad(+) <==> 5,10-methylene-h(4)folate + co(2) + nh(3) + nadh + h(+)the glycine cleavage system is widely distributed in animals, plants and bacteria and consists of three intrinsic and one common components: those are i) p-protein, a pyridoxal phosphate-containing protein, ii) t-protein, a protein required for the tetrahydrofolate-dependent reaction, iii) h-protein, a protein that carries the aminomet ...200818941301
ruva and ruvb mutants specifically impaired for replication fork reversal.replication fork reversal (rfr) is a reaction that takes place in escherichia coli at replication forks arrested by the inactivation of a replication protein. fork reversal involves the annealing of the leading and lagging strand ends; it results in the formation of a holliday junction adjacent to dna double-strand end, both of which are processed by recombination enzymes. in several replication mutants, replication fork reversal is catalysed by the ruvab complex, originally characterized for it ...200818942176
side-chain recognition and gating in the ribosome exit tunnel.the ribosome is a large complex catalyst responsible for the synthesis of new proteins, an essential function for life. new proteins emerge from the ribosome through an exit tunnel as nascent polypeptide chains. recent findings indicate that tunnel interactions with the nascent polypeptide chain might be relevant for the regulation of translation. however, the specific ribosomal structural features that mediate this process are unknown. performing molecular dynamics simulations, we are studying ...200818946046
revealing unique properties of the ribosome using a network based analysis.the ribosome is a complex molecular machine that offers many potential sites for functional interference, therefore representing a major target for antibacterial drugs. the growing number of high-resolution structures of ribosomes from different organisms, in free form and in complex with various ligands, provides unique data for structural and comparative analyses of rna structures. we model the ribosome structure as a network, where nucleotides are represented as nodes and intermolecular inter ...200818625614
conserved discrimination against misacylated trnas by two mesophilic elongation factor tu orthologs.elongation factor tu (ef-tu) binds and loads elongating aminoacyl-trnas (aa-trnas) onto the ribosome for protein biosynthesis. many bacteria biosynthesize gln-trna (gln) and asn-trna (asn) by an indirect, two-step pathway that relies on the misacylated trnas glu-trna (gln) and asp-trna (asn) as intermediates. previous thermodynamic and experimental analyses have demonstrated that thermus thermophilus ef-tu does not bind asp-trna (asn) and predicted a similar discriminatory response against glu-t ...200818627126
the complete genome sequence of moorella thermoacetica (f. clostridium thermoaceticum).this paper describes the genome sequence of moorella thermoacetica (f. clostridium thermoaceticum), which is the model acetogenic bacterium that has been widely used for elucidating the wood-ljungdahl pathway of co and co(2) fixation. this pathway, which is also known as the reductive acetyl-coa pathway, allows acetogenic (often called homoacetogenic) bacteria to convert glucose stoichiometrically into 3 mol of acetate and to grow autotrophically using h(2) and co as electron donors and co(2) as ...200818631365
molecular mechanisms underlying the positive stringent response of the bacillus subtilis ilv-leu operon, involved in the biosynthesis of branched-chain amino acids.branched-chain amino acids are the most abundant amino acids in proteins. the bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. this operon exhibits a rela-dependent positive stringent response to amino acid starvation. we investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. deletion analysis involving various lacz fusions revealed two molecular mechanisms underlying the positive stringent response of ...200818641142
erythromycin-induced ribosome stalling and rnase j1-mediated mrna processing in bacillus subtilis.summary: addition of erythromycin (em) to a bacillus subtilis strain carrying the ermc gene results in ribosome stalling in the ermc leader peptide coding sequence. using deltaermc, a deletion derivative of ermc that specifies the 254 nucleotide deltaermc mrna, we showed previously that ribosome stalling is concomitant with processing of deltaermc mrna, generating a 209 nucleotide rna whose 5' end maps to codon 5 of the deltaermc coding sequence. here we probed for peptidyl-trna to show that rib ...200818647167
the highest affinity binding site of small protein b on transfer messenger rna is outside the trna domain.eubacterial ribosomes stalled on defective mrnas are released through a mechanism referred to as trans-translation, depending on the coordinated actions of small protein b (smpb) and transfer messenger rna (tmrna). a series of tmrna variants with deletions in each structural domain were produced. their structures were monitored by enzymatic and chemical probes in vitro, in the presence and absence of smpb. dissociation constants between these rnas and smpb from aquifex aeolicus were derived by s ...200818648069
a new regulatory circuit in ribosomal protein operons: s2-mediated control of the rpsb-tsf expression in vivo.autogenous regulation is a general strategy of balancing ribosomal protein synthesis in bacteria. control mechanisms have been studied in detail for most of ribosomal protein operons, except for rpsb-tsf encoding essential r-protein s2 and elongation factor ts, where even the promoter has remained unknown. by using single-copy translational fusions with the chromosomal lacz gene and western-blot analysis, we demonstrate here that s2 serves as a negative regulator of both rpsb and tsf expression ...200818648071
crenarchaeal arginine decarboxylase evolved from an s-adenosylmethionine decarboxylase enzyme.the crenarchaeon sulfolobus solfataricus uses arginine to produce putrescine for polyamine biosynthesis. however, genome sequences from s. solfataricus and most crenarchaea have no known homologs of the previously characterized pyridoxal 5'-phosphate or pyruvoyl-dependent arginine decarboxylases that catalyze the first step in this pathway. instead they have two paralogs of the s-adenosylmethionine decarboxylase (adometdc). the gene at locus sso0585 produces an adometdc enzyme, whereas the gene ...200818650422
structure and mechanistic implications of a uroporphyrinogen iii synthase-product complex.uroporphyrinogen iii synthase (u3s) catalyzes the asymmetrical cyclization of a linear tetrapyrrole to form the physiologically relevant uroporphyrinogen iii (uro'gen iii) isomer during heme biosynthesis. here, we report four apoenzyme and one product complex crystal structures of the thermus thermophilus (hb27) u3s protein. the overlay of eight crystallographically unique u3s molecules reveals a huge range of conformational flexibility, including a "closed" product complex. the product, uro'gen ...200818651750
distinct double- and single-stranded dna binding of e. coli replicative dna polymerase iii alpha subunit.the alpha subunit of the replicative dna polymerase iii of escherichia coli is the active polymerase of the 10-subunit bacterial replicase. the c-terminal region of the alpha subunit is predicted to contain an oligonucleotide binding (ob-fold) domain. in a series of optical tweezers experiments, the alpha subunit is shown to have an affinity for both double- and single-stranded dna, in distinct subdomains of the protein. the portion of the protein that binds to double-stranded dna stabilizes the ...200818652472
origin of the nucleus and ran-dependent transport to safeguard ribosome biogenesis in a chimeric cell.the origin of the nucleus is a central problem about the origin of eukaryotes. the common ancestry of nuclear pore complexes (npc) and vesicle coating complexes indicates that the nucleus evolved via the modification of a pre-existing endomembrane system. such an autogenous scenario is cell biologically feasible, but it is not clear what were the selective or neutral mechanisms that had led to the origin of the nuclear compartment.200818652645
a unique conformation of the anticodon stem-loop is associated with the capacity of trnafmet to initiate protein synthesis.in all organisms, translational initiation takes place on the small ribosomal subunit and two classes of methionine trna are present. the initiator is used exclusively for initiation of protein synthesis while the elongator is used for inserting methionine internally in the nascent polypeptide chain. the crystal structure of escherichia coli initiator trna(f)(met) has been solved at 3.1 a resolution. the anticodon region is well-defined and reveals a unique structure, which has not been describe ...200818653533
recr forms a ring-like tetramer that encircles dsdna by forming a complex with recf.in the recfor pathway, the recf and recr proteins form a complex that binds to dna and exerts multiple functions, including directing the loading of reca onto single-stranded (ss) dna regions near double-stranded (ds) dna-ssdna junctions and preventing it from forming a filament beyond the ssdna region. however, neither the structure of the recfr complex nor its dna-binding mechanism was previously identified. here, size-exclusion chromatography and small-angle x-ray scattering data indicate tha ...200818658243
crystal structure of the thermus thermophilus 16 s rrna methyltransferase rsmc in complex with cofactor and substrate guanosine.post-transcriptional modification is a ubiquitous feature of ribosomal rna in all kingdoms of life. modified nucleotides are generally clustered in functionally important regions of the ribosome, but the functional contribution to protein synthesis is not well understood. here we describe high resolution crystal structures for the n(2)-guanine methyltransferase rsmc that modifies residue g1207 in 16 s rrna near the decoding site of the 30 s ribosomal subunit. rsmc is a class i s-adenosyl-l-methi ...200818667428
the rna acetyltransferase driven by atp hydrolysis synthesizes n4-acetylcytidine of trna anticodon.the wobble base of escherichia coli elongator trna(met) is modified to n(4)-acetylcytidine (ac(4)c), which is thought to ensure the precise recognition of the aug codon by preventing misreading of near-cognate aua codon. by employing genome-wide screen of uncharacterized genes in escherichia coli ('ribonucleome analysis'), we found the ypfi gene, which we named tmca (trna(met) cytidine acetyltransferase), to be responsible for ac(4)c formation. tmca is an enzyme that contains a walker-type atpas ...200818668122
thermodynamic redox behavior of the heme centers in a-type heme-copper oxygen reductases: comparison between the two subfamilies.the study of the thermodynamic redox behavior of the hemes from two members of the a family of heme-copper oxygen reductases, paracoccus denitrificans aa3 (a1 subfamily) and rhodothermus marinus caa3 (a2 subfamily) enzymes, is presented. at different ph values, midpoint reduction potentials and interaction potentials were obtained in the framework of a pairwise model for two interacting redox centers. in both enzymes, the hemes have different reduction potentials. for the a1-type enzyme, it was ...200818676644
deciphering the genetic determinants for aerobic nicotinic acid degradation: the nic cluster from pseudomonas putida kt2440.the aerobic catabolism of nicotinic acid (na) is considered a model system for degradation of n-heterocyclic aromatic compounds, some of which are major environmental pollutants; however, the complete set of genes as well as the structural-functional relationships of most of the enzymes involved in this process are still unknown. we have characterized a gene cluster (nic genes) from pseudomonas putida kt2440 responsible for the aerobic na degradation in this bacterium and when expressed in heter ...200818678916
cloning, expression, purification and preliminary crystallographic analysis of the rnase hi domain of the mycobacterium tuberculosis protein rv2228c as a maltose-binding protein fusion.the predicted ribonuclease (rnase) hi domain of the open reading frame rv2228c from mycobacterium tuberculosis has been cloned as a hexahistidine fusion and a maltose-binding protein (mbp) fusion. expression was only observed for the mbp-fusion protein, which was purified using amylose affinity chromatography and gel filtration. the rnase hi domain could be cleaved from the mbp-fusion protein by factor xa digestion, but was very unstable. in contrast, the fusion protein was stable, could be obta ...200818678948
structure of the e. coli dna glycosylase alka bound to the ends of duplex dna: a system for the structure determination of lesion-containing dna.the constant attack on dna by endogenous and exogenous agents gives rise to nucleobase modifications that cause mutations, which can lead to cancer. visualizing the effects of these lesions on the structure of duplex dna is key to understanding their biologic consequences. the most definitive method of obtaining such structures, x-ray crystallography, is troublesome to employ owing to the difficulty of obtaining diffraction-quality crystals of dna. here, we present a crystallization system that ...200818682218
hyperthermophilic aquifex aeolicus initiates primer synthesis on a limited set of trinucleotides comprised of cytosines and guanines.the placement of the extreme thermophile aquifex aeolicus in the bacterial phylogenetic tree has evoked much controversy. we investigated whether adaptations for growth at high temperatures would alter a key functional component of the replication machinery, specifically dnag primase. although the structure of bacterial primases is conserved, the trinucleotide initiation specificity for a. aeolicus was hypothesized to differ from other microbes as an adaptation to a geothermal milieu. to determi ...200818684998
the growth-promoting and stress response activities of the bacillus subtilis gtp binding protein obg are separable by mutation.bacillus subtilis obg is a ribosome-associating gtp binding protein that is needed for growth, sporulation, and induction of the bacterium's general stress regulon (gsr). it is unclear whether the roles of obg in sporulation and stress responsiveness are direct or a secondary effect of its growth-promoting functions. the present work addresses this question by an analysis of two obg alleles whose phenotypes argue for direct roles for obg in each process. the first allele [obg(g92d)] encodes a mi ...200818689482
insights into the replisome from the structure of a ternary complex of the dna polymerase iii alpha-subunit.the crystal structure of the catalytic alpha-subunit of the dna polymerase iii (pol iiialpha) holoenzyme bound to primer-template dna and an incoming deoxy-nucleoside 5'-triphosphate has been determined at 4.6-a resolution. the polymerase interacts with the sugar-phosphate backbone of the dna across its minor groove, which is made possible by significant movements of the thumb, finger, and beta-binding domains relative to their orientations in the unliganded polymerase structure. additionally, t ...200818691598
crystal structure of type 2 isopentenyl diphosphate isomerase from thermus thermophilus in complex with inorganic pyrophosphate.the n-terminal region is stabilized in the crystal structure of thermus thermophilus type 2 isopentenyl diphosphate isomerase in complex with inorganic pyrophosphate, providing new insights about the active site and the catalytic mechanism of the enzyme. the pp i moiety is located near the conserved residues, h10, r97, h152, q157, e158, and w219, and the flavin cofactor. the putative active site of isopentenyl diphosphate isomerase 2 provides interactions for stabilizing a carbocationic intermed ...200818693754
crystal structures of nadh:fmn oxidoreductase (emob) at different stages of catalysis.edta has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. recently, two critical enzymes, edta monooxygenase (emoa) and nadh:fmn oxidoreductase (emob), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the edta degradation pathway in mesorhizobium sp. bnc1. emoa is an fmnh2-dependent enzyme that requires emob to provide fmnh2 for the conversion of edta to ethylenediaminedia ...200818701448
mitochondrial nadh fluorescence is enhanced by complex i binding.mitochondrial nadh fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. mitochondrial nadh fluorescence is enhanced several-fold in the matrix through extended fluorescence lifetimes (efl). however, the actual binding sites responsible for nadh efl are unknown. we tested the hypothesis that nadh binding to complex i is a significant source of mitochondrial nadh fluorescence enhancement. to test this hypothesis, the effect of complex i binding on n ...200818702505
genetic identification of yeast 18s rrna residues required for efficient recruitment of initiator trna(met) and aug selection.high-resolution structures of bacterial 70s ribosomes have provided atomic details about mrna and trna binding to the decoding center during elongation, but such information is lacking for preinitiation complexes (pics). we identified residues in yeast 18s rrna critical in vivo for recruiting methionyl trna(i)(met) to 40s subunits during initiation by isolating mutations that derepress gcn4 mrna translation. several such gcd(-) mutations alter the a928:u1389 base pair in helix 28 (h28) and allow ...200818708582
slr1923 of synechocystis sp. pcc6803 is essential for conversion of 3,8-divinyl(proto)chlorophyll(ide) to 3-monovinyl(proto)chlorophyll(ide).the deduced amino acid sequence of an slr1923 gene of synechocystis sp. pcc6803 is homologous to archaean f(420)h(2) dehydrogenase, which acts as a soluble subcomplex of reduced nicotinamide adenine dinucleotide dehydrogenase complex i. in this study, the gene was inactivated and characteristics of the mutant were analyzed. the mutant grew slower than the wild type under 100 microe m(-2) s(-1) but did not grow under high light intensity (300 microe m(-2) s(-1)). the cellular content of chlorophy ...200818715956
s-adenosyl-l-methionine hydrolase (adenosine-forming), a conserved bacterial and archaeal protein related to sam-dependent halogenases. 200818720493
purification and characterization of the bacterial udp-glcnac:undecaprenyl-phosphate glcnac-1-phosphate transferase weca.to date, the structural and functional characterization of proteins belonging to the polyprenyl-phosphate n-acetylhexosamine-1-phosphate transferase superfamily has been relentlessly held back by problems encountered with their overexpression and purification. in the present work and for the first time, the integral membrane protein weca that catalyzes the transfer of the glcnac-1-phosphate moiety from udp-glcnac onto the carrier lipid undecaprenyl phosphate, yielding undecaprenyl-pyrophosphoryl ...200818723618
the rate and character of spontaneous mutation in thermus thermophilus.selection of spontaneous, loss-of-function mutations at two chromosomal loci (pyrf and pyre) enabled the first molecular-level analysis of replication fidelity in the extremely thermophilic bacterium thermus thermophilus. two different methods yielded similar mutation rates, and mutational spectra determined by sequencing of independent mutants revealed a variety of replication errors distributed throughout the target genes. the genomic mutation rate estimated from these targets, 0.00097 +/- 0.0 ...200818723895
the d subunit plays a central role in human vacuolar h(+)-atpases.the multi-subunit vacuolar-type h(+)-atpase consists of a v(1) domain (a-h subunits) catalyzing atp hydrolysis and a v(0) domain (a, c, c', c", d, e) responsible for h(+) translocation. the mammalian v(0) d subunit is one of the least-well characterized, and its function and position within the pump are still unclear. it has two different forms encoded by separate genes, d1 being ubiquitous while d2 is predominantly expressed at the cell surface in kidney and osteoclast. to determine whether it ...200818752060
electron and proton transfer in the ba(3) oxidase from thermus thermophilus.the ba(3)-type cytochrome c oxidase from thermus thermophilus is phylogenetically very distant from the aa(3)-type cytochrome c oxidases. nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of o(2) to water is catalysed at a haem a(3)-cu(b) catalytic site. the three-dimensional structure of the ba(3) oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, rhodobacter sphaeroide ...200818752061
structure of the guide-strand-containing argonaute silencing complex.the slicer activity of the rna-induced silencing complex is associated with argonaute, the rnase h-like piwi domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger rna. here we report on the crystal structure of thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base dna guide strand, thereby identifying the nucleic-acid-binding channel positioned between the paz- and piwi-containing lobes, as well as the pivot-like conformational changes assoc ...200818754009
ybea is the m3psi methyltransferase rlmh that targets nucleotide 1915 in 23s rrna.pseudouridines in the stable rnas of bacteria are seldom subjected to further modification. there are 11 pseudouridine (psi) sites in escherichia coli rrna, and further modification is found only at psi1915 in 23s rrna, where the n-3 position of the base becomes methylated. here, we report the identity of the e. coli methyltransferase that specifically catalyzes methyl group addition to form m(3)psi1915. analyses of e. coli rrnas using maldi mass spectrometry showed that inactivation of the ybea ...200818755835
the oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect trna positioning.the oxazolidinones represent the first new class of antibiotics to enter into clinical usage within the past 30 years, but their binding site and mechanism of action has not been fully characterized. we have determined the crystal structure of the oxazolidinone linezolid bound to the deinococcus radiodurans 50s ribosomal subunit. linezolid binds in the a site pocket at the peptidyltransferase center of the ribosome overlapping the aminoacyl moiety of an a-site bound trna as well as many clinical ...200818757750
mechanism of cu(a) assembly.copper is essential for proper functioning of cytochrome c oxidases, and therefore for cellular respiration in eukaryotes and many bacteria. here we show that a new periplasmic protein (pcu(a)c) selectively inserts cu(i) ions into subunit ii of thermus thermophilus ba(3) oxidase to generate a native cu(a) site. the purported metallochaperone sco1 is unable to deliver copper ions; instead, it works as a thiol-disulfide reductase to maintain the correct oxidation state of the cu(a) cysteine ligand ...200818758441
crystal structure of escherichia coli rnk, a new rna polymerase-interacting protein.sequence-based searches identified a new family of genes in proteobacteria, named rnk, which shares high sequence similarity with the c-terminal domains of the gre factors (grea and greb) and the thermus/deinococcus anti-gre factor gfh1. we solved the x-ray crystal structure of escherichia coli regulator of nucleoside kinase (rnk) at 1.9 a resolution using the anomalous signal from the native protein. the rnk structure strikingly resembles those of e. coli grea and greb and thermus gfh1, all of ...200818760284
methanogen homoaconitase catalyzes both hydrolyase reactions in coenzyme b biosynthesis.homoaconitase enzymes catalyze hydrolyase reactions in the alpha-aminoadipate pathway for lysine biosynthesis or the 2-oxosuberate pathway for methanogenic coenzyme b biosynthesis. despite the homology of this iron-sulfur protein to aconitase, previously studied homoaconitases catalyze only the hydration of cis-homoaconitate to form homoisocitrate rather than the complete isomerization of homocitrate to homoisocitrate. the mj1003 and mj1271 proteins from the methanogen methanocaldococcus jannasc ...200818765671
transplantation of a tyrosine editing domain into a tyrosyl-trna synthetase variant enhances its specificity for a tyrosine analog.to guarantee specific trna and amino acid pairing, several aminoacyl-trna synthetases correct aminoacylation errors by deacylating or "editing" misaminoacylated trna. a previously developed variant of escherichia coli tyrosyl-trna synthetase (iodotyrrs) esterifies or "charges" trna(tyr) with a nonnatural amino acid, 3-iodo-l-tyrosine, and with l-tyrosine less efficiently. in the present study, the editing domain of phenylalanyl-trna synthetase (phers) was transplanted into iodotyrrs to edit tyro ...200818765802
development of trna synthetases and connection to genetic code and disease.the genetic code is established by the aminoacylation reactions of aminoacyl trna synthetases, where amino acids are matched with triplet anticodons imbedded in the cognate trnas. the code established in this way is so robust that it gave birth to the entire tree of life. the trna synthetases are organized into two classes, based on their active site architectures. the details of this organization, and other considerations, suggest how the synthetases evolved by gene duplications, and how early ...200818765819
crystal structure of a sulfur carrier protein complex found in the cysteine biosynthetic pathway of mycobacterium tuberculosis.the structure of the protein complex cysm-cyso from a new cysteine biosynthetic pathway found in the h37rv strain of mycobacterium tuberculosis has been determined at 1.53 a resolution. cysm (rv1336) is a plp-containing beta-replacement enzyme and cyso (rv1335) is a sulfur carrier protein with a ubiquitin-like fold. cysm catalyzes the replacement of the acetyl group of o-acetylserine by cyso thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reactio ...200818771296
full-length escherichia coli seca dimerizes in a closed conformation in solution as determined by cryo-electron microscopy.seca is an obligatory component of the escherichia coli general secretion pathway. however, the oligomeric structure of seca and seca conformational changes during translocation processes are still unclear. here we obtained the three-dimensional structure of e. coli wild-type full-length seca in solution by single particle cryo-electron microscopy and determined its oligomeric organization. in this structure, seca occurs as a dimer in which the two protomers are arranged in an antiparallel mode, ...200818772144
mechanism of the chemical step for the guanosine triphosphate (gtp) hydrolysis catalyzed by elongation factor tu.elongation factor tu (ef-tu), the protein responsible for delivering aminoacyl-trnas (aa-trnas) to ribosomal a site during translation, belongs to the group of guanosine-nucleotide (gtp/gdp) binding proteins. its active 'on'-state corresponds to the gtp-bound form, while the inactive 'off'-state corresponds to the gdp-bound form. in this work we focus on the chemical step, gtp+h(2)o-->gdp+pi, of the hydrolysis mechanism. we apply molecular modeling tools including molecular dynamics simulations ...200818773979
inhibition of a transcriptional pause by rna anchoring to rna polymerase.we describe a mechanism by which nascent rna inhibits transcriptional pausing. putl rna of bacteriophage hk022 suppresses transcription termination at downstream terminators and pausing within a nearby u-rich sequence. in vitro transcription and footprinting assays reveal that this pausing results from backtracking of rna polymerase and that binding of nascent putl rna to polymerase limits backtracking by restricting re-entry of the transcript into the rna exit channel. the restriction is local ...200818775328
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