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an unusual mechanism of bacterial gene expression revealed for the rnase p protein of thermus strains.the rnase p protein gene (rnpa) completely overlaps the rpmh gene (encoding ribosomal protein l34) out of frame in the thermophilic bacterium thermus thermophilus. this results in the synthesis of an extended rnase p protein (c5) of 163 aa and, by inference, of 240 aa in the related strain thermus filiformis. start codons of rnpa and rpmh, apparently governed by the same ribosome binding site, are separated by only 4 nt, which suggests a regulatory linkage between l34 and c5 translation and, acc ...200312719542
expanding trna recognition of a trna synthetase by a single amino acid change.aspartyl-trna synthetase (asprs) occurs in two types: the discriminating enzyme (d-asprs) forms only asp-trna(asp), whereas the nondiscriminating enzyme (nd-asprs) also synthesizes asp-trna(asn), which is a required intermediate for protein synthesis in many organisms. we attempted to expand the trna recognition of the discriminating thermococcus kodakaraensis asprs to that of a nd-asprs by in vitro mutagenesis. an alignment of 26 archaeal asprs proteins revealed two positions (26 and 85 in the ...200312730374
global expression profiling and physiological characterization of corynebacterium glutamicum grown in the presence of l-valine.addition of l-valine (50 to 200 mm) to glucose minimal medium had no effect on the growth of wild-type corynebacterium glutamicum atcc 13032 but inhibited the growth of the derived valine production strain val1 [13032 deltailva deltapanbc(pjc1ilvbncd)] in a concentration-dependent manner. in order to explore this strain-specific valine effect, genomewide expression profiling was performed using dna microarrays, which showed that valine caused an increased ilvbn mrna level in val1 but not in the ...200312732517
rna polymerase mutations that impair conversion to a termination-resistant complex by q antiterminator proteins.bacteriophage lambda q-protein stably binds and modifies rna polymerase (rnap) to a termination-resistant form. we describe amino acid substitutions in rnap that disrupt q-mediated antitermination in vivo and in vitro. the positions of these substitutions in the modeled rnap/dna/rna ternary elongation complex, and their biochemical properties, suggest that they do not define a binding site for q in rnap, but instead act by impairing interactions among core rnap subunits and nucleic acids that ar ...200312756229
an intersubunit contact stimulating transcription initiation by e coli rna polymerase: interaction of the alpha c-terminal domain and sigma region 4.the c-terminal domain of the escherichia coli rna polymerase (rnap) alpha subunit (alphactd) stimulates transcription initiation by interacting with upstream (up) element dna and a variety of transcription activators. here we identify specific substitutions in region 4.2 of sigma 70 (sigma(70)) and in alphactd that decrease transcription initiation from promoters containing some, but not all, up elements. this decrease in transcription derives from a decrease in the initial equilibrium constant ...200312756230
crystal structure of trna(m1g37)methyltransferase: insights into trna recognition.trna(m(1)g37)methyltransferase (trmd) catalyzes the transfer of a methyl group from s-adenosyl-l- methionine (adomet) to g(37) within a subset of bacterial trna species, which have a g residue at the 36th position. the modified guanosine is adjacent to and 3' of the anticodon and is essential for the maintenance of the correct reading frame during translation. here we report four crystal structures of trmd from haemophilus influenzae, as binary complexes with either adomet or s-adenosyl-l-homocy ...200312773376
protein synthesis in escherichia coli with mischarged trna.two types of aspartyl-trna synthetase exist: the discriminating enzyme (d-asprs) forms only asp-trna(asp), while the nondiscriminating one (nd-asprs) also synthesizes asp-trna(asn), a required intermediate in protein synthesis in many organisms (but not in escherichia coli). on the basis of the e. coli trpa34 missense mutant transformed with heterologous nd-asps genes, we developed a system with which to measure the in vivo formation of asp-trna(asn) and its acceptance by elongation factor ef-tu ...200312775689
nmr structure of the active conformation of the varkud satellite ribozyme cleavage site.substrate cleavage by the neurospora varkud satellite (vs) ribozyme involves a structural change in the stem-loop i substrate from an inactive to an active conformation. we have determined the nmr solution structure of a mutant stem-loop i that mimics the active conformation of the cleavage site internal loop. this structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. the active internal loop displays differ ...200312782785
functional asymmetry in the lysyl-trna synthetase explored by molecular dynamics, free energy calculations and experiment.charging of transfer-rna with cognate amino acid is accomplished by the aminoacyl-trna synthetases, and proceeds through an aminoacyl adenylate intermediate. the lysyl-trna synthetase has evolved an active site that specifically binds lysine and atp. previous molecular dynamics simulations of the heat-inducible escherichia coli lysyl-trna synthetase, lysu, have revealed differences in the binding of atp and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme ...200312787471
the bacterium thermus thermophilus, like hyperthermophilic archaea, uses a two-step pathway for the synthesis of mannosylglycerate.the biosynthetic pathway for the synthesis of the compatible solute alpha-mannosylglycerate (mg) in the thermophilic bacterium thermus thermophilus hb27 was identified based on the activities of recombinant mannosyl-3-phosphoglycerate synthase (mpgs) (ec 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (mpgp) (ec 3.1.3.70). the sequences of homologous genes from the archaeon pyrococcus horikoshii were used to identify mpgs and mpgp genes in t. thermophilus hb27 genome. both genes were sepa ...200312788726
quantitation of human immunodeficiency virus type 1 in breast milk.the distribution and stability of human immunodeficiency virus type 1 (hiv-1) in breast milk (bm) components remain largely unknown. inhibitory effects, if any, of bm on hiv rna and dna pcr amplification are poorly understood. we have addressed these issues by using virus-spiked bm samples from hiv-negative women. bm samples from hiv-negative women were spiked with hiv-1 virions or cells containing a single integrated copy of hiv dna (8e5/lav). after incubation under different experimental condi ...200312791866
a snorna that guides the two most conserved pseudouridine modifications within rrna confers a growth advantage in yeast.ribosomal rnas contain a number of modified nucleotides. the most abundant nucleotide modifications found within rrnas fall into two types: 2'-o-ribose methylations and pseudouridylations. in eukaryotes, small nucleolar guide rnas, the snornas that are the rna components of the snornps, specify the position of these modifications. the 2'-o-ribose methylations and pseudouridylations are guided by the box c/d and box h/aca snornas, respectively. the role of these modifications in rrna remains poor ...200312810910
viridans group streptococci are donors in horizontal transfer of topoisomerase iv genes to streptococcus pneumoniae.a total of 46 ciprofloxacin-resistant (cip(r)) streptococcus pneumoniae strains were isolated from 1991 to 2001 at the hospital of bellvitge. five of these strains showed unexpectedly high rates of nucleotide variations in the quinolone resistance-determining regions (qrdrs) of their parc, pare, and gyra genes. the nucleotide sequence of the full-length parc, pare, and gyra genes of one of these isolates revealed a mosaic structure compatible with an interspecific recombination origin. southern ...200312821449
dissection of the triple tryptophan electron transfer chain in escherichia coli dna photolyase: trp382 is the primary donor in photoactivation.in escherichia coli photolyase, excitation of the fad cofactor in its semireduced radical state (fadh*) induces an electron transfer over approximately 15 a from tryptophan w306 to the flavin. it has been suggested that two additional tryptophans are involved in an electron transfer chain fadh* <-- w382 <-- w359 <-- w306. to test this hypothesis, we have mutated w382 into redox inert phenylalanine. ultrafast transient absorption studies showed that, in wt photolyase, excited fadh* decayed with a ...200312835419
contribution of structural genomics to understanding the biology of escherichia coli. 200312837772
determinants of the src homology domain 3-like fold.in eukaryotes, the src homology domain 3 (sh3) is a very important motif in signal transduction. sh3 domains recognize poly-proline-rich peptides and are involved in protein-protein interactions. until now, the existence of sh3 domains has not been demonstrated in prokaryotes. however, the structure of the c-terminal domain of dtxr clearly shows that the fold of this domain is very similar to that of the sh3 domain. in addition, there is evidence that the c-terminal domain of dtxr binds to poly- ...200312837782
crystal structures of the quinone oxidoreductase from thermus thermophilus hb8 and its complex with nadph: implication for nadph and substrate recognition.the crystal structures of the zeta-crystalline-like soluble quinone oxidoreductase from thermus thermophilus hb8 (qor(tt)) and of its complex with nadph have been determined at 2.3- and 2.8-a resolutions, respectively. qor(tt) is composed of two domains, and its overall fold is similar to the folds of escherichia coli quinone oxidoreductase (qor(ec)) and horse liver alcohol dehydrogenase. qor(tt) forms a homodimer in the crystal by interaction of the betaf-strands in domain ii, forming a large b ...200312837796
structural basis for thermostability of beta-glycosidase from the thermophilic eubacterium thermus nonproteolyticus hg102.the three-dimensional structure of a thermostable beta-glycosidase (gly(tn)) from the thermophilic eubacterium thermus nonproteolyticus hg102 was determined at a resolution of 2.4 a. the core of the structure adopts the (betaalpha)(8) barrel fold. the sequence alignments and the positions of the two glu residues in the active center indicate that gly(tn) belongs to the glycosyl hydrolases of retaining family 1. we have analyzed the structural features of gly(tn) related to the thermostability an ...200312837801
detecting protein-protein interactions in the intact cell of bacillus subtilis (atcc 6633).the salt bridge, paired group-specific reagent cyanogen (ethanedinitrile; c(2)n(2)) converts naturally occurring pairs of functional groups into covalently linked products. cyanogen readily permeates cell walls and membranes. when the paired groups are shared between associated proteins, isolation of the covalently linked proteins allows their identity to be assigned. examination of organisms of known genome sequence permits identification of the linked proteins by mass spectrometric techniques ...200312837803
pilin-like proteins in the extremely thermophilic bacterium thermus thermophilus hb27: implication in competence for natural transformation and links to type iv pilus biogenesis.the extreme thermophile thermus thermophilus hb27 exhibits high frequencies of natural transformation. although we recently reported identification of the first competence genes in thermus, the molecular basis of dna uptake is unknown. a pilus-like structure is assumed to be involved. twelve genes encoding prepilin-like proteins were identified in three loci in the genome of t. thermophilus. mutational analyses, described in this paper, revealed that one locus, which contains four genes that enc ...200312839734
a modified saccharomyces cerevisiae strain that consumes l-arabinose and produces ethanol.metabolic engineering is a powerful method to improve, redirect, or generate new metabolic reactions or whole pathways in microorganisms. here we describe the engineering of a saccharomyces cerevisiae strain able to utilize the pentose sugar l-arabinose for growth and to ferment it to ethanol. expanding the substrate fermentation range of s. cerevisiae to include pentoses is important for the utilization of this yeast in economically feasible biomass-to-ethanol fermentation processes. after over ...200312839792
development of antibiotic-overproducing strains by site-directed mutagenesis of the rpsl gene in streptomyces lividans.certain rpsl (which encodes the ribosomal protein s12) mutations that confer resistance to streptomycin markedly activate the production of antibiotics in streptomyces spp. these rpsl mutations are known to be located in the two conserved regions within the s12 protein. to understand the roles of these two regions in the activation of silent genes, we used site-directed mutagenesis to generate eight novel mutations in addition to an already known (k88e) mutation that is capable of activating ant ...200312839808
molecular phylogenetic exploration of bacterial diversity in a bakreshwar (india) hot spring and culture of shewanella-related thermophiles.the bacterial diversity of a hot spring in bakreshwar, india, was investigated by a culture-independent approach. 16s ribosomal dna clones derived from the sediment samples were found to be associated with gamma-proteobacteria, cyanobacteria, and green nonsulfur and low-gc gram-positive bacteria. the first of the above phylotypes cobranches with shewanella, a well-known iron reducer. this phylogenetic correlation has been exploited to develop culture conditions for thermophilic iron-reducing mic ...200312839826
direct observation of protonation reactions during the catalytic cycle of cytochrome c oxidase.cytochrome c oxidase, the terminal protein in the respiratory chain, converts oxygen into water and helps generate the electrochemical gradient used in the synthesis of atp. the catalytic action of cytochrome c oxidase involves electron transfer, proton transfer, and o2 reduction. these events trigger specific molecular changes at the active site, which, in turn, influence changes throughout the protein, including alterations of amino acid side chain orientations, hydrogen bond patterns, and pro ...200312851460
x-ray crystal structures of the wt and a hyper-accurate ribosome from escherichia coli.protein biosynthesis on the ribosome requires accurate reading of the genetic code in mrna. two conformational rearrangements in the small ribosomal subunit, a closing of the head and body around the incoming trna and an rna helical switch near the mrna decoding site, have been proposed to select for complementary base-pairing between mrna codons and trna anticodons. we determined x-ray crystal structures of the wt and a hyper-accurate variant of the escherichia coli ribosome at resolutions of 1 ...200312853578
a new thermus sp. class-iis enzyme sub-family: isolation of a 'twin' endonuclease tspdti with a novel specificity 5'-atgaa(n(11/9))-3', related to tspgwi, taqii and tth111ii.the tspdti restriction endonuclease, which shows a novel recognition specificity 5'-atgaa(n(11/9))-3', was isolated from thermus sp. dt. tspdti appears to be a 'twin' of restriction endonuclease tspgwi from thermus sp. gw, as we have previously reported. tspgwi was isolated from the same location as tspdti, it recognizes a related sequence 5'-acgga(n(11/9))-3' and has conserved cleavage positions. both enzymes resemble two other class-iis endonucleases from thermus sp.: taqii and tth111ii. n-ter ...200312853651
ribosomal localization of translation initiation factor if2.bacterial translation initiation factor if2 is a gtp-binding protein that catalyzes binding of initiator fmet-trna in the ribosomal p site. the topographical localization of if2 on the ribosomal subunits, a prerequisite for understanding the mechanism of initiation complex formation, has remained elusive. here, we present a model for the positioning of if2 in the 70s initiation complex as determined by cleavage of rrna by the chemical nucleases cu(ii):1,10-orthophenanthroline and fe(ii):edta tet ...200312869707
when contemporary aminoacyl-trna synthetases invent their cognate amino acid metabolism.faithful protein synthesis relies on a family of essential enzymes called aminoacyl-trna synthetases, assembled in a piecewise fashion. analysis of the completed archaeal genomes reveals that all archaea that possess asparaginyl-trna synthetase (asnrs) also display a second orf encoding an asnrs truncated from its anticodon binding-domain (asnrs2). we show herein that pyrococcus abyssi asnrs2, in contrast to asnrs, does not sustain asparaginyl-trnaasn synthesis but is instead capable of converti ...200312874385
identification of protein biochemical functions by similarity search using the molecular surface database ef-site.the identification of protein biochemical functions based on their three-dimensional structures is strongly required in the post-genome-sequencing era. we have developed a new method to identify and predict protein biochemical functions using the similarity information of molecular surface geometries and electrostatic potentials on the surfaces. our prediction system consists of a similarity search method based on a clique search algorithm and the molecular surface database ef-site (electrostati ...200312876308
crystal structure of the conserved protein tt1542 from thermus thermophilus hb8.the tt1542 protein from thermus thermophilus hb8 is annotated as a conserved hypothetical protein, and belongs to the duf158 family in the pfam database. a blast search revealed that homologs of tt1542 are present in a wide range of organisms. the tt1542 homologs in eukaryotes, pig-l in mammals, and gpi12 in yeast and protozoa, have n-acetylglucosaminylphosphatidylinositol (glcnac-pi) de-n-acetylase activity. although most of the homologs in prokaryotes are hypothetical and have no known functio ...200312876312
dynamic reorganization of the functionally active ribosome explored by normal mode analysis and cryo-electron microscopy.combining structural data for the ribosome from x-ray crystallography and cryo-electron microscopy with dynamic models based on elastic network normal mode analysis, an atomically detailed picture of functionally important structural rearrangements that occur during translocation is elucidated. the dynamic model provides a near-atomic description of the ratchet-like rearrangement of the 70s ribosome seen in cryo-electron microscopy, and permits the identification of bridging interactions that ei ...200312878726
a novel is element, is621, of the is110/is492 family transposes to a specific site in repetitive extragenic palindromic sequences in escherichia coli.an escherichia coli strain, ecor28, was found to have insertions of an identical sequence (1,279 bp in length) at 10 loci in its genome. this insertion sequence (named is621) has one large open reading frame encoding a putative protein that is 326 amino acids in length. a computer-aided homology search using the dna sequence as the query revealed that is621 was homologous to the piv genes, encoding pilin gene invertase (piv). a homology search using the amino acid sequence of the putative protei ...200312897009
temperature-dependent hypermutational phenotype in reca mutants of thermus thermophilus hb27.the reca gene from thermus thermophilus hb27 was cloned and engineered to obtain insertion (reca::kat) and deletion (deltareca) derivatives. transcription of reca in this extreme thermophile was induced by mitomycin c, leading to the synthesis of a monocistronic mrna. this dna damage-mediated induction was dependent on the integrity of reca. in addition to uv sensitivity, the reca mutants of t. thermophilus showed severe pleiotropic defects, ranging from irregular nucleoid condensation and segre ...200312897010
directed evolution of thermus maltogenic amylase toward enhanced thermal resistance.the thermostability of maltogenic amylase from thermus sp. strain im6501 (thma) was improved greatly by random mutagenesis using dna shuffling. four rounds of dna shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme thma-dm, which had a total of seven individual mutations. the seven amino acid substitutions in thma-dm were identified as r26q, s169n, i333v, m375t, a398v, q411l, and p453l. the optimal reaction temperature of the recombinant enzyme ...200312902281
is the in situ accessibility of the 16s rrna of escherichia coli for cy3-labeled oligonucleotide probes predicted by a three-dimensional structure model of the 30s ribosomal subunit?systematic studies on the hybridization of fluorescently labeled, rrna-targeted oligonucleotides have shown strong variations in in situ accessibility. reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. during the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. these studies ...200312902289
comparative genomics of bacterial zinc regulons: enhanced ion transport, pathogenesis, and rearrangement of ribosomal proteins.zinc is an important component of many proteins, but in large concentrations it is poisonous to the cell. thus its transport is regulated by zinc repressors zur of proteobacteria and gram-positive bacteria from the bacillus group and adcr of bacteria from the streptococcus group. comparative computational analysis allowed us to identify binding signals of zur repressors gaaatgttatantataacatttc for gamma-proteobacteria, gtaatgtaataacattac for the agrobacterium group, gatatgttataacatatc for the rh ...200312904577
glutathione synthetase homologs encode alpha-l-glutamate ligases for methanogenic coenzyme f420 and tetrahydrosarcinapterin biosyntheses.proteins in the atp-grasp superfamily of amide bond-forming ligases have evolved to function in a number of unrelated biosynthetic pathways. previously identified homologs encoding glutathione synthetase, d-alanine:d-alanine ligase and the bacterial ribosomal protein s6:glutamate ligase have been vertically inherited within certain organismal lineages. although members of this specificity-diverse superfamily share a common reaction mechanism, the nonoverlapping set of amino acid and peptide subs ...200312909715
analysis of the dna joining repertoire of chlorella virus dna ligase and a new crystal structure of the ligase-adenylate intermediate.chlorella virus dna ligase is the smallest eukaryotic atp-dependent dna ligase known; it suffices for yeast cell growth in lieu of the essential yeast dna ligase cdc9. the chlorella virus ligase-adenylate intermediate has an intrinsic nick sensing function and its dna footprint extends 8-9 nt on the 3'-hydroxyl (3'-oh) side of the nick and 11-12 nt on the 5'-phosphate (5'-po4) side. here we establish the minimal length requirements for ligatable 3'-oh and 5'-po4 strands at the nick (6 nt) and de ...200312930960
mutations in rpobc suppress the defects of a sinorhizobium meliloti rela mutant.the nitrogen-fixing symbiosis between sinorhizobium meliloti and medicago sativa requires complex physiological adaptation by both partners. one method by which bacteria coordinately control physiological adaptation is the stringent response, which is triggered by the presence of the nucleotide guanosine tetraphosphate (ppgpp). ppgpp, produced by the rela enzyme, is thought to bind to and alter the ability of rna polymerase (rnap) to initiate and elongate transcription and affect the affinity of ...200312949113
inference of protein function and protein linkages in mycobacterium tuberculosis based on prokaryotic genome organization: a combined computational approach.the genome of mycobacterium tuberculosis was analyzed using recently developed computational approaches to infer protein function and protein linkages. we evaluated and employed a method to infer genes likely to belong to the same operon, as judged by the nucleotide distance between genes in the same genomic orientation, and combined this method with those of the rosetta stone, phylogenetic profile and conserved gene neighbor computational methods for the inference of protein function.200312952538
characterization of microbial communities in gas industry pipelines.culture-independent techniques, denaturing gradient gel electrophoresis (dgge) analysis, and random cloning of 16s rrna gene sequences amplified from community dna were used to determine the diversity of microbial communities in gas industry pipelines. samples obtained from natural gas pipelines were used directly for dna extraction, inoculated into sulfate-reducing bacterium medium, or used to inoculate a reactor that simulated a natural gas pipeline environment. the variable v2-v3 (average siz ...200312957923
molecular analysis of echovirus 13 isolates and aseptic meningitis, spain.echovirus 13 (ev13), considered rare, was reported worldwide in 2000, mostly related to aseptic meningitis outbreaks. in spain, 135 ev13 isolates were identified. the genetic relationships between 64 representative strains from spain and other reported isolates from the united states, germany, italy, japan, and sweden were described by analyzing the partial sequence of the major capsid protein (vp1) gene. the strains from spain were clearly identified as ev13 (79.5% similarity with the ev13 refe ...200312967490
single-nucleotide polymorphism genotyping on optical thin-film biosensor chips.single-nucleotide polymorphisms (snps) constitute the bulk of human genetic variation and provide excellent markers to identify genetic factors contributing to complex disease susceptibility. a rapid, sensitive, and inexpensive assay is important for large-scale snp scoring. here we report the development of a multiplex snp detection system using silicon chips coated to create a thin-film optical biosensor. allele-discriminating, aldehyde-labeled oligonucleotides are arrayed and covalently attac ...200312975525
the extended loops of ribosomal proteins l4 and l22 are not required for ribosome assembly or l4-mediated autogenous control.ribosomal proteins l4 and l22 both have a globular domain that sits on the surface of the large ribosomal subunit and an extended loop that penetrates its core. the tips of both loops contribute to the lining of the peptide exit tunnel and have been implicated in a gating mechanism that might regulate the exit of nascent peptides. also, the extensions of l4 and l22 contact multiple domains of 23s rrna, suggesting they might facilitate rrna folding during ribosome assembly. to learn more about th ...200313130133
a noncognate aminoacyl-trna synthetase that may resolve a missing link in protein evolution.efforts to delineate the advent of many enzymes essential to protein translation are often limited by the fact that the modern genetic code evolved before divergence of the tree of life. glutaminyl-trna synthetase (glnrs) is one noteworthy exception to the universality of the translation apparatus. in eukaryotes and some bacteria, this enzyme is essential for the biosynthesis of gln-trnagln, an obligate intermediate in translation. glnrs is absent, however, in archaea, and most bacteria, organel ...200313679580
role of residual structure in the unfolded state of a thermophilic protein.ribonucleases h from the thermophilic bacterium thermus thermophilus and the mesophile escherichia coli demonstrate a dramatic and surprising difference in their change in heat capacity upon unfolding (deltacp degrees ). the lower deltacp degrees of the thermophilic protein directly contributes to its higher thermal denaturation temperature (tm). we propose that this deltacp degrees difference originates from residual structure in the unfolded state of the thermophilic protein; we verify this hy ...200314504401
genetic characterization of optochin-susceptible viridans group streptococci.two clinical isolates of viridans group streptococci (vs) with different degrees of susceptibility to optochin (opt), i.e., fully opt-susceptible (opt(s)) vs strain 1162/99 (for which the mic was equal to that for streptococcus pneumoniae, 0.75 micro g/ml) and intermediate opt(s) vs strain 1174/97 (mic, 6 micro g/ml) were studied. besides being opt susceptible, they showed characteristics typical of vs, such as bile insolubility; lack of reaction with pneumococcal capsular antibodies; and lack o ...200314506029
osmotic adaptation of thermus thermophilus rq-1: lesson from a mutant deficient in synthesis of trehalose.strains of thermus thermophilus accumulate primarily trehalose and smaller amounts of mannosylglycerate in response to salt stress in yeast extract-containing media (o. c. nunes, c. m. manaia, m. s. da costa, and h. santos, appl. environ. microbiol. 61:2351-2357, 1995). a 2.4-kbp dna fragment from t. thermophilus strain rq-1 carrying otsa (encoding trehalose-phosphate synthase [tps]), otsb (encoding trehalose-phosphate phosphatase [tpp]), and a short sequence of the 5' end of tres (trehalose syn ...200314526004
atp-bound conformation of topoisomerase iv: a possible target for quinolones in streptococcus pneumoniae.topoisomerase iv, a c(2)e(2) tetramer, is involved in the topological changes of dna during replication. this enzyme is the target of antibacterial compounds, such as the coumarins, which target the atp binding site in the pare subunit, and the quinolones, which bind, outside the active site, to the quinolone resistance-determining region (qrdr). after site-directed and random mutagenesis, we found some mutations in the atp binding site of pare near the dimeric interface and outside the qrdr tha ...200314526026
the ribosome and yidc. new insights into the biogenesis of escherichia coli inner membrane proteins.in the bacterium escherichia coli, inner membrane proteins (imps) are generally targeted through the signal recognition particle pathway to the sec translocon, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. lateral transport seems to be assisted by the imp yidc. in this article, we discuss recent observations that point to a key role for the ribosome in imp targeting and to the diverse roles of yidc in imp assembly.200314528263
structures of deacylated trna mimics bound to the e site of the large ribosomal subunit.during translation, trnas cycle through three binding sites on the ribosome: the a, the p, and the e sites. we have determined the structures of complexes between the haloarcula marismortui large ribosomal subunit and two different e site substrates: a deacylated trna acceptor stem minihelix and a cca-acceptor end. both of these trna mimics contain analogs of adenosine 76, the component responsible for a large proportion of e site binding affinity. they bind in the center of the loop-extension o ...200314561884
the saccharomyces cerevisiae u2 snrna:pseudouridine-synthase pus7p is a novel multisite-multisubstrate rna:psi-synthase also acting on trnas.the saccharomyces cerevisiae pus7 protein was recently characterized as a novel rna:pseudouridine (psi)-synthase acting at position 35 in u2 snrna. however, u2 snrna was the only potential substrate tested for this enzyme. in this work, we demonstrated that although pus7p is responsible for the formation of only one of the six psi residues present in yeast usnrnas, it catalyzes u to psi conversion at position 13 in cytoplasmic trnas and at position 35 in pre-trna(tyr). sites of rna modification ...200314561887
microbial community structure in midgut and hindgut of the humus-feeding larva of pachnoda ephippiata (coleoptera: scarabaeidae).the guts of soil-feeding macroinvertebrates contain a complex microbial community that is involved in the transformation of ingested soil organic matter. in a companion paper (t. lemke, u. stingl, m. egert, m. w. friedrich, and a. brune, appl. environ. microbiol. 69:6650-6658, 2003), we show that the gut of our model organism, the humivorous larva of the cetoniid beetle pachnoda ephippiata, is characterized by strong midgut alkalinity, high concentrations of microbial fermentation products, and ...200314602626
analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification.we analyzed the tuf gene, encoding elongation factor tu, from 33 strains representing 17 lactobacillus species and 8 bifidobacterium species. the tuf sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. we demonstrated that the synonymous substitution affecting this gene renders elongation factor tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. in fact, the phylogeny generated by these tu ...200314602655
comparative analysis of editosome proteins in trypanosomatids.detailed comparisons of 16 editosome proteins from trypanosoma brucei, trypanosoma cruzi and leishmania major identified protein motifs associated with catalysis and protein or nucleic acid interactions that suggest their functions in rna editing. five related proteins with rnase iii-like motifs also contain a u1-like zinc finger and either dsrbm or pumilio motifs. these proteins may provide the endoribonuclease function in editing. two other related proteins, at least one of which is associated ...200314602897
enhancement of dna, cdna synthesis and fidelity at high temperatures by a dimeric single-stranded dna-binding protein.bacterial single-stranded dna-binding proteins (ssbs) are required for dna replication and repair. we have over-expressed and purified the native form and two his-tagged fusions of the ssb from thermus thermophilus (tthssb). the three proteins were found as dimers in solution. they bound in vitro to single-stranded dna specifically over a temperature range of 4-80 degrees c, and the wild-type protein could withstand incubation at 94 degrees c for 2 min. addition of tthssb to pcr halved the elong ...200314602905
j protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix.the major hsp70 of the mitochondrial matrix (ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix. tim44, a peripheral inner membrane protein with limited sequence similarity to the j domain of j-type cochaperones, tethers ssc1 to the import channel. here we report that, unlike a j protein, tim44 does not stimulate the atpase activity of ssc1, nor does it affect the stimulation by either a known mi ...200314605210
coevolution of an aminoacyl-trna synthetase with its trna substrates.glutamyl-trna synthetases (glurss) occur in two types, the discriminating and the nondiscriminating enzymes. they differ in their choice of substrates and use either trnaglu or both trnaglu and trnagln. although most organisms encode only one glurs, a number of bacteria encode two different glurs proteins; yet, the trna specificity of these enzymes and the reason for such gene duplications are unknown. a database search revealed duplicated glurs genes in >20 bacterial species, suggesting that th ...200314615592
products transcribed from rearranged rrn genes of escherichia coli can assemble to form functional ribosomes.to examine the flexibility of rrna operons with respect to fundamental organization, transcription, processing, and assembly of ribosomes, operon variations were introduced by a plasmid into an escherichia coli strain that has deletions of all chromosomal copies of rrna genes. in the reconstructed operons, a salmonella intervening sequence (ivs) from 23s helix 45 was introduced into the e. coli 23s gene at the same position. three different constructs of the e. coli 16s gene were then placed who ...200314617656
quantification of alternatively spliced fgfr2 rnas using the rna invasive cleavage assay.the regulated splicing of fibroblast growth factor receptor-2 (fgfr2) transcripts leads to tissue-specific expression of distinct receptor isoforms. these isoforms contain two different versions of the ligand binding ig-like domain iii, which are encoded by exon iiib or exon iiic. the mutually exclusive use of exon iiib and exon iiic can be recapitulated in tissue culture using dt3 and at3 rat prostate carcinoma cells. we used this well-characterized system to evaluate the precision and accuracy ...200314624010
solution nmr structure of the 30s ribosomal protein s28e from pyrococcus horikoshii.we report nmr assignments and solution structure of the 71-residue 30s ribosomal protein s28e from the archaean pyrococcus horikoshii, target jr19 of the northeast structural genomics consortium. the structure, determined rapidly with the aid of automated backbone resonance assignment (autoassign) and automated structure determination (autostructure) software, is characterized by a four-stranded beta-sheet with a classic greek-key topology and an oligonucleotide/oligosaccharide beta-barrel (ob) ...200314627742
an archaebacteria-derived glutamyl-trna synthetase and trna pair for unnatural amino acid mutagenesis of proteins in escherichia coli.the addition of novel amino acids to the genetic code of escherichia coli involves the generation of an aminoacyl-trna synthetase and trna pair that is 'orthogonal', meaning that it functions independently of the synthetases and trnas endogenous to e.coli. the amino acid specificity of the orthogonal synthetase is then modified to charge the corresponding orthogonal trna with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon. ...200314627803
regulation of lysine biosynthesis and transport genes in bacteria: yet another rna riboswitch?comparative analysis of genes, operons and regulatory elements was applied to the lysine biosynthetic pathway in available bacterial genomes. we report identification of a lysine-specific rna element, named the lys element, in the regulatory regions of bacterial genes involved in biosynthesis and transport of lysine. similarly to the previously described rna regulatory elements for three vitamins (riboflavin, thiamin and cobalamin), purine and methionine regulons, this regulatory rna structure i ...200314627808
ribosomal proteins rps0 and rps21 of saccharomyces cerevisiae have overlapping functions in the maturation of the 3' end of 18s rrna.the rps0 proteins of saccharomyces cerevisiae are components of the 40s ribosomal subunit required for maturation of the 3' end of 18s rrna. drosophila and human homologs of the rps0 proteins physically interact with rps21 proteins, and decreased expression of both proteins in drosophila impairs control of cellular proliferation in hematopoietic organs during larval development. here, we characterize the yeast rps21a/b genes and show that strains where both genes are disrupted are not viable. re ...200314627813
transcript cleavage factors grea and greb act as transient catalytic components of rna polymerase.prokaryotic transcription elongation factors grea and greb stimulate intrinsic nucleolytic activity of rna polymerase (rnap). the proposed biological role of gre-induced rna hydrolysis includes transcription proofreading, suppression of transcriptional pausing and arrest, and facilitation of rnap transition from transcription initiation to transcription elongation. using an array of biochemical and molecular genetic methods, we mapped the interaction interface between gre and rnap and identified ...200314633991
effect of g-1 on histidine trna microhelix conformation.histidine trnas (trna(his)) are unique in that they possess an extra 5'-base (g-1) not found in other trnas. deletion of g-1 results in at least a 250-fold reduction in the rate of histidine charging in vitro. to better understand the role of the g-1 nucleotide in defining the structure of trna(his), and to correlate structure with cognate amino acid charging, nmr and molecular dynamics (md) studies were performed on the wild-type and a deltag-1 mutant escherichia coli histidine trna acceptor st ...200314654706
crystal structures that suggest late development of genetic code components for differentiating aromatic side chains.early forms of the genetic code likely generated "statistical" proteins, with similar side chains occupying the same sequence positions at different ratios. in this scenario, groups of related side chains were treated by aminoacyl-trna synthetases as a single molecular species until a discrimination mechanism developed that could separate them. the aromatic amino acids tryptophan, tyrosine, and phenylalanine likely constituted one of these groups. a crystal structure of human tryptophanyl-trna s ...200314671330
structure of mth11/mth rpp29, an essential protein subunit of archaeal and eukaryotic rnase p.we have determined the solution structure of mth11 (mth rpp29), an essential subunit of the rnase p enzyme from the archaebacterium methanothermobacter thermoautotrophicus (mth). rnase p is a ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving the 5' leader sequence during maturation of trnas in all three domains of life. in eubacteria, this enzyme is made up of two subunits: a large rna ( approximately 120 kda) responsible for mediating catalysis, and a small protein cofactor ...200314673079
grpe, a nucleotide exchange factor for dnak.the cochaperone grpe functions as a nucleotide exchange factor to promote dissociation of adenosine 5'-diphosphate (adp) from the nucleotide-binding cleft of dnak. grpe and the dnaj cochaperone act in concert to control the flux of unfolded polypeptides into and out of the substrate-binding domain of dnak by regulating the nucleotide-bound state of dnak. dnaj stimulates nucleotide hydrolysis, and grpe promotes the exchange of adp for adenosine triphosphate (atp) and also augments peptide release ...200314984054
structure of the topoisomerase vi-b subunit: implications for type ii topoisomerase mechanism and evolution.type iia and type iib topoisomerases each possess the ability to pass one dna duplex through another in an atp-dependent manner. the role of atp in the strand passage reaction is poorly understood, particularly for the type iib (topoisomerase vi) family. we have solved the structure of the atp-binding subunit of topoisomerase vi (topovi-b) in two states: an unliganded monomer and a nucleotide-bound dimer. we find that topovi-b is highly structurally homologous to the entire 40-43 kda atpase regi ...200312505993
role of ctc from listeria monocytogenes in osmotolerance.listeria monocytogenes is a food-borne pathogen with the ability to grow under conditions of high osmolarity. in a previous study, we reported the identification of 12 proteins showing high induction after salt stress. one of these proteins is highly similar to the general stress protein ctc of bacillus subtilis. in this study, induction of ctc after salt stress was confirmed at the transcriptional level by using rna slot blot experiments. to explore the role of the ctc gene product in resistanc ...200312513990
nucleic acid recognition by ob-fold proteins.the ob-fold domain is a compact structural motif frequently used for nucleic acid recognition. structural comparison of all ob-fold/nucleic acid complexes solved to date confirms the low degree of sequence similarity among members of this family while highlighting several structural sequence determinants common to most of these ob-folds. loops connecting the secondary structural elements in the ob-fold core are extremely variable in length and in functional detail. however, certain features of l ...200312598368
extremophiles 2002. 200312813059
atom depth as a descriptor of the protein interior.atom depth, defined as the distance (dpx, a) of a nonhydrogen atom from its closest solvent-accessible protein neighbor, provides a simple but precise description of the protein interior. mean residue depths can be easily computed and are very sensitive to structural features. from the analysis of the average and maximum atom depths of a set of 136 protein structures, we derive a limit of approximately 200 residues for protein and protein domain size. the average and maximum atom depths in a pro ...200312668463
deep trefoil knot implicated in rna binding found in an archaebacterial protein. 200312486711
toward an understanding of the structural basis of translation.the recently solved x-ray crystal structures of the ribosome have provided opportunities for studying the molecular basis of translation with a variety of methods including cryo-electron microscopy - where maps give the first glimpses of ribosomal evolution - and fluorescence spectroscopy techniques.200314659007
position of eukaryotic initiation factor eif1 on the 40s ribosomal subunit determined by directed hydroxyl radical probing.eukaryotic initiation factor (eif) eif1 maintains the fidelity of initiation codon selection by enabling 43s complexes to reject codon-anticodon mismatches, to recognize the initiation codon context, and to discriminate against establishing a codon-anticodon interaction with augs located <8 nt from the 5'-end of mrna. to understand how eif1 plays its discriminatory role, we determined its position on the 40s ribosomal subunit using directed hydroxyl radical cleavage. the cleavage of 18s rrna in ...200314600024
solvation change and ion release during aminoacylation by aminoacyl-trna synthetases.discrimination between cognate and non-cognate trnas by aminoacyl-trna synthetases occurs at several steps of the aminoacylation pathway. we have measured changes of solvation and counter-ion distribution at various steps of the aminoacylation pathway of glutamyl- and glutaminyl-trna synthetases. the decrease in the association constant with increasing kcl concentration is relatively small for cognate trna binding when compared to known dna-protein interactions. the electro-neutral nature of the ...200314530451
over-expression of interleukin 10 in mucosal t cells of patients with active ulcerative colitis.ulcerative colitis (uc), a chronic inflammatory bowel disease, exhibits pronounced increase of t lymphocytes in the inflamed mucosa. to understand the role of intestinal t lymphocytes in the pathogenesis of uc their cytokine production in the mucosa was analysed. intestinal t lymphocytes of uc, crohn's disease and control patients were analysed for cytokine mrna levels by real-time quantitative reverse transcription-polymerase chain reaction (rt-pcr) directly after isolation without in vitro sti ...200312974765
biochemical characterization of the kink-turn rna motif.rna, which acts as a medium for transmitting genetic information, plays a variety of roles in a cell. as with proteins, elucidation of the three- dimensional (3d) structures of rnas is important for understanding their various roles. determination of the atomic structures of crystallized ribosome has enabled the identification of previously unknown rna structural motifs. the kink-turn (k-turn or ga) motif, which causes a sharp bend in an rna double helix, was identified as one of these structura ...200314500816
rna structure comparison, motif search and discovery using a reduced representation of rna conformational space.given the wealth of new rna structures and the growing list of rna functions in biology, it is of great interest to understand the repertoire of rna folding motifs. the ability to identify new and known motifs within novel rna structures, to compare tertiary structures with one another and to quantify the characteristics of a given rna motif are major goals in the field of rna research; however, there are few systematic ways to address these issues. using a novel approach for visualizing and mat ...200312907716
trna synthetase paralogs: evolutionary links in the transition from trna-dependent amino acid biosynthesis to de novo biosynthesis. 200312913115
structural classification of zinc fingers: survey and summary.zinc fingers are small protein domains in which zinc plays a structural role contributing to the stability of the domain. zinc fingers are structurally diverse and are present among proteins that perform a broad range of functions in various cellular processes, such as replication and repair, transcription and translation, metabolism and signaling, cell proliferation and apoptosis. zinc fingers typically function as interaction modules and bind to a wide variety of compounds, such as nucleic aci ...200312527760
interstice mutations that block site-to-site translocation of a misactivated amino acid bound to a class i trna synthetase.class i aminoacyl-trna synthetases catalyze editing reactions that prevent ambiguity from entering the genetic code. misactivated amino acids are translocated in cis from the active site for aminoacylation to the center for editing, located approximately 30 a away. mutational analysis has functionally separated the two sites by creating mutations that disrupt the catalytic center for editing but not for aminoacylation and vice versa. what is not known is whether translocation per se can be disru ...200312515858
pops: a fast algorithm for solvent accessible surface areas at atomic and residue level.pops (parameter optimsed surfaces) is a new method to calculate solvent accessible surface areas, which is based on an empirically parameterisable analytical formula and fast to compute. atomic and residue areas (the latter represented by a single sphere centered on the c(alpha) atom of amino acids and at the p atom of nucleotides) have been optimised versus accurate all-atom methods. the parameterisation has been derived from a selected dataset of proteins and nucleic acids of different sizes a ...200312824328
the long-range electrostatic interactions control trna-aminoacyl-trna synthetase complex formation.in most cases aminoacyl-trna synthetases (aarss) are negatively charged, as are the trna substrates. it is apparent that there are driving forces that provide a long-range attraction between like charge aars and trna, and ensure formation of "close encounters." based on numerical solutions to the nonlinear poisson-boltzmann equation, we evaluated the electrostatic potential generated by different aarss. the 3d-isopotential surfaces calculated for different aarss at 0.01 kt/e contour level reveal ...200312761395
unified two-metal mechanism of rna synthesis and degradation by rna polymerase.in dna-dependent rna polymerases, reactions of rna synthesis and degradation are performed by the same active center (in contrast to dna polymerases in which they are separate). we propose a unified catalytic mechanism for multisubunit rna polymerases based on the analysis of its 3'-5' exonuclease reaction in the context of crystal structure. the active center involves a symmetrical pair of mg(2+) ions that switch roles in synthesis and degradation. one ion is retained permanently and the other ...200312727889
chloroplast elongation factor ts pro-protein is an evolutionarily conserved fusion with the s1 domain-containing plastid-specific ribosomal protein-7.the components of chloroplast translation are similar to those of prokaryotic translation but contain some additional unique features. proteomic analysis of the chlamydomonas reinhardtii chloroplast ribosome identified an s1-like protein, plastid-specific ribosomal protein-7 (psrp-7), as a stoichiometric component of the 30s subunit. here, we report that psrp-7 is part of a polyprotein that contains psrp-7 on its amino end and two translation elongation factor ts (ef-ts) domains at the carboxy e ...200415548736
shear rate moderates community diversity in freshwater biofilms.the development of freshwater multispecies biofilms at solid-liquid interfaces occurs both in quiescent waters and under conditions of high shear rates. however, the influence of hydrodynamic shear rates on bacterial biofilm diversity is poorly understood. we hypothesized that different shear rates would significantly influence biofilm diversity and alter the relative proportions of coaggregating and autoaggregating community isolates. in order to study this hypothesis, freshwater biofilms were ...200415574945
bayesfold: rational 2 degrees folds that combine thermodynamic, covariation, and chemical data for aligned rna sequences.bayesfold is a web application that folds an alignment of closely related sequences and evaluates hypotheses about their shared structure. it uses bayes's theorem to combine information from several sources, including chemical mapping (if available), thermodynamic folding, and observed sequence variations. its method provides a rational basis for integrating results, even when these methods conflict. on a gapped alignment of 86 trnaphe sequences each 77 bases long, bayesfold takes 31 sec to perf ...200415317972
structure of the photolyase-like domain of cryptochrome 1 from arabidopsis thaliana.signals generated by cryptochrome (cry) blue-light photoreceptors are responsible for a variety of developmental and circadian responses in plants. the crys are also identified as circadian blue-light photoreceptors in drosophila and components of the mammalian circadian clock. these flavoproteins all have an n-terminal domain that is similar to photolyase, and most have an additional c-terminal domain of variable length. we present here the crystal structure of the photolyase-like domain of cry ...200415299148
three-dimensional motifs from the scor, structural classification of rna database: extruded strands, base triples, tetraloops and u-turns.release 2.0.1 of the structural classification of rna (scor) database, http://scor.lbl.gov, contains a classification of the internal and hairpin loops in a comprehensive collection of 497 nmr and x-ray rna structures. this report discusses findings of the classification that have not been reported previously. the scor database contains multiple examples of a newly described rna motif, the extruded helical single strand. internal loop base triples are classified in scor according to their three- ...200415121895
structural basis of the interaction between the aaa atpase p97/vcp and its adaptor protein p47.the aaa atpase p97/vcp is involved in many cellular events including ubiquitin-dependent processes and membrane fusion. in the latter, the p97 adaptor protein p47 is of central importance. in order to provide insight into the molecular basis of p97 adaptor binding, we have determined the crystal structure of p97 nd1 domains complexed with p47 c-terminal domain at 2.9 a resolution. the structure reveals that the p47 ubiquitin regulatory x domain (ubx) domain interacts with the p97 n domain via a ...200414988733
the kink-turn motif in rna is dimorphic, and metal ion-dependent.the kink-turn (k-turn) is a new motif in rna structure that was identified by examination of the crystal structures of the ribosome. we examined the structural and dynamic properties of this element in free solution. the k-turn rna exists in a dynamic equilibrium between a tightly kinked conformation and a more open structure similar to a simple bulge bend. the highly kinked form is stabilized by the noncooperative binding of metal ions, but a significant population of the less-kinked form is pr ...200414730024
4scopmap: automated assignment of protein structures to evolutionary superfamilies.inference of remote homology between proteins is very challenging and remains a prerogative of an expert. thus a significant drawback to the use of evolutionary-based protein structure classifications is the difficulty in assigning new proteins to unique positions in the classification scheme with automatic methods. to address this issue, we have developed an algorithm to map protein domains to an existing structural classification scheme and have applied it to the scop database.200415598351
correspondence between anomalous m- and deltacp-values in protein folding.proteins folding according to a classical two-state system characteristically show v-shaped chevron plots. we have previously interpreted the symmetrically curved chevron plot of the protein u1a as denaturant-dependent movements in the position of the transition state ensemble (tse). s6, a structural analog of u1a, shows a classical v-shaped chevron plot indicative of straightforward two-state kinetics, but the mutant la30 has a curved unfolding limb, which is most consistent with tse mobility. ...200415557266
the driving force for molecular evolution of translation.it is widely argued that protein synthesis evolved out of an rna world, in which catalytic and other biological functions now carried out by proteins were performed by rnas. however, it is not clear what selective advantage would have provided the driving force for evolution of a primitive translation apparatus, because of the unlikelihood that rudimentary polypeptides would have contributed sufficiently useful biological functions. here, i suggest that the availability of even simple peptides c ...200415547132
application of sparse nmr restraints to large-scale protein structure prediction.the protein structure prediction algorithm touchstonex that uses sparse distance restraints derived from nmr nuclear overhauser enhancement (noe) data to predict protein structures at low-to-medium resolution was evaluated as follows: first, a representative benchmark set of the protein data bank library consisting of 1365 proteins up to 200 residues was employed. using n/8 simulated long-range restraints, where n is the number of residues, 1023 (75%) proteins were folded to a c(alpha) root-mean ...200415298926
sequence-specific cleavage of small-subunit (ssu) rrna with oligonucleotides and rnase h: a rapid and simple approach to ssu rrna-based quantitative detection of microorganisms.a rapid and simple approach to the small-subunit (ssu) rrna-based quantitative detection of a specific group of microorganisms in complex ecosystems has been developed. the method employs sequence-specific cleavage of rrna molecules with oligonucleotides and rnase h. defined mixtures of ssu rrnas were mixed with an oligonucleotide (referred to as a "scissor probe") that was specifically designed to hybridize with a particular site of targeted rrna and were subsequently digested with rnase h to p ...200415184170
the identification of novel rna structural motifs using compadres: an automated approach to structural discovery.recurring rna structural motifs are important sites of tertiary interaction and as such, are integral to rna macromolecular structure. although numerous rna motifs have been classified and characterized, the identification of new motifs is of great interest. in this study, we discovered four new conformationally recurring motifs: the pi-turn, the omega-turn, the alpha-loop and the c2'-endo mediated flipped adenosine motif. not only do they have complex and interesting structures, but they partic ...200415608296
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