Publications
| Title | Abstract | Year(sorted descending) Filter | PMID Filter |
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| interactions among cii protein, rna polymerase and the lambda pre promoter: contacts between rna polymerase and the -35 region of pre are identical in the presence and absence of cii protein. | the dna recognition sequence for the transcriptional activator, cii protein, which is critical for lysogenization by bacteriophage lambda, overlaps the -35 region of the p(re) promoter. data presented here show that activation by cii does not change the pattern of cleavage of the -35 region of p(re) by iron (s)-1-(p-bromoacetamidobenzyl)-edta (fe-babe) conjugated to the sigma subunit of rna polymerase (rnap). thus, the overall interaction between sigma and the -35 region of p(re) is not signific ... | 2004 | 14872063 |
| atypical archaeal trna pyrrolysine transcript behaves towards ef-tu as a typical elongator trna. | the newly discovered trna(pyl) is involved in specific incorporation of pyrrolysine in the active site of methylamine methyltransferases in the archaeon methanosarcina barkeri. in solution probing experiments, a transcript derived from trna(pyl) displays a secondary fold slightly different from the canonical cloverleaf and interestingly similar to that of bovine mitochondrial trna(ser)(uga). aminoacylation of trna(pyl) transcript by a typical class ii synthetase, lysrs from yeast, was possible w ... | 2004 | 14872064 |
| mycobacterium tuberculosis rv2118c codes for a single-component homotetrameric m1a58 trna methyltransferase. | modified nucleosides in trnas play important roles in trna structure, biosynthesis and function, and serve as crucial determinants of bacterial growth and virulence. in the yeast saccharomyces cerevisiae, mutants defective in n1-methylation of a highly conserved adenosine (a58) in the tpsic loop of initiator trna are non-viable. the yeast m1a58 methyltransferase is a heterotetramer consisting of two different polypeptide chains, gcd14p and gcd10p. interestingly, while m1a58 is not found in most ... | 2004 | 14960715 |
| functional group recognition at the aminoacylation and editing sites of e. coli valyl-trna synthetase. | to correct misactivation and misacylation errors, escherichia coli valyl-trna synthetase (valrs) catalyzes a trna(val)-dependent editing reaction at a site distinct from its aminoacylation site. here we examined the effects of replacing the conserved 3'-adenosine of trna(val) with nucleoside analogs, to identify structural elements of the 3'-terminal nucleoside necessary for trna function at the aminoacylation and editing sites of valrs. the results show that the exocyclic amino group (n6) is no ... | 2004 | 14970394 |
| domain movements of elongation factor eef2 and the eukaryotic 80s ribosome facilitate trna translocation. | an 11.7-a-resolution cryo-em map of the yeast 80s.eef2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eef2 and the 80s ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. sordarin positions domain iii of eef2 so that it can interact with the sarcin-ricin loop of 25s rrna and protein rps23 (s12p). this particular conformation explains the inhibitory action of sordari ... | 2004 | 14976550 |
| dominant gain-of-function mutations in hsp104p reveal crucial roles for the middle region. | heat-shock protein 104 (hsp104p) is a protein-remodeling factor that promotes survival after extreme stress by disassembling aggregated proteins and can either promote or prevent the propagation of prions (protein-based genetic elements). hsp104p can be greatly overexpressed without slowing growth, suggesting tight control of its powerful protein-remodeling activities. we isolated point mutations in hsp104p that interfere with this control and block cell growth. each mutant contained alterations ... | 2004 | 14978213 |
| differential recruitment of nuclear receptor coactivators may determine alternative rna splice site choice in target genes. | the biological consequences of steroid hormone-mediated transcriptional activation of target genes might be difficult to predict because alternative splicing of a single neosynthesized precursor rna can result in production of different protein isoforms with opposite biological activities. therefore, an important question to address is the manner in which steroid hormones affect the splicing of their target gene transcripts. in this report, we demonstrate that individual steroid hormones had dif ... | 2004 | 14982999 |
| efficiency and pattern of uv pulse laser-induced rna-rna cross-linking in the ribosome. | escherichia coli ribosomes were irradiated with a krf excimer laser (248 nm, 22 ns pulse) with incident pulse energies in the range of 10-40 mj for a 1 cm2 area, corresponding to fluences of 4.5 to 18 x 10(9) w m(-2), to determine strand breakage yields and the frequency and pattern of rna-rna cross- linking in the 16s rrna. samples were irradiated in a cuvette with one laser pulse or in a flow cell with an average of 4.6 pulses per sample. the yield of strand breaks per photon was intensity dep ... | 2004 | 14999094 |
| crystal structure of rlmai: implications for understanding the 23s rrna g745/g748-methylation at the macrolide antibiotic-binding site. | the rlma class of enzymes (rlma(i) and rlma(ii)) catalyzes n1-methylation of a guanine base (g745 in gram-negative and g748 in gram-positive bacteria) of hairpin 35 of 23s rrna. we have determined the crystal structure of escherichia coli rlma(i) at 2.8-a resolution, providing 3d structure information for the rlma class of rna methyltransferases. the dimeric protein structure exhibits features that provide new insights into its molecular function. each rlma(i) molecule has a zn-binding domain, r ... | 2004 | 14999102 |
| definition of bases in 23s rrna essential for ribosomal subunit association. | the ribosome is a two-subunit molecular machine, sporting a working cycle that involves coordinated movements of the subunits. recent structural studies of the 70s ribosome describe a rather large number of intersubunit contacts, some of which are dynamic during translocation. we set out to determine which intersubunit contacts are functionally indispensable for the association of ribosome subunits by using a modification interference approach. modification of the n-1 position of a715, a1912, or ... | 2004 | 15037769 |
| probing the q-proton pathway of ba3-cytochrome c oxidase by time-resolved fourier transform infrared spectroscopy. | in cytochrome c oxidase, the terminal respiratory enzyme, electron transfers are strongly coupled to proton movements within the enzyme. two proton pathways (k and d) containing water molecules and hydrophobic amino acids have been identified and suggested to be involved in the proton translocation from the mitochondrial matrix or the bacterial cytoplasm into the active site. in addition to the k and d proton pathways, a third proton pathway (q) has been identified only in ba3-cytochrome c oxida ... | 2004 | 15041681 |
| dna damage recognition of mutated forms of uvrb proteins in nucleotide excision repair. | the dna repair protein uvrb plays an indispensable role in the stepwise and sequential damage recognition of nucleotide excision repair in escherichia coli. our previous studies suggested that uvrb is responsible for the chemical damage recognition only upon a strand opening mediated by uvra. difficulties were encountered in studying the direct interaction of uvrb with adducts due to the presence of uvra. we report herein that a single point mutation of y95w in which a tyrosine is replaced by a ... | 2004 | 15065863 |
| thermodynamic characterization of the interaction of mutant uvrb protein with damaged dna. | during the dna damage recognition of nucleotide excision repair in escherichia coli the interaction of uvrb protein with damaged dna ensures the recognition of differences in the intrinsic chemical structures of a variety of adduct molecules in dna double helix. our earlier study indicated that a single tyrosine-to-tryptophan mutation at residue 95 converted the uvrb to a protein [uvrb(y95w)] that is able to bind to a structure-specific bubble dna substrate, even in the absence of uvra. fluoresc ... | 2004 | 15065864 |
| an aminoacyl-trna synthetase-like protein encoded by the escherichia coli yadb gene glutamylates specifically trnaasp. | the product of the escherichia coli yadb gene is homologous to the n-terminal part of bacterial glutamyl-trna synthetases (glurss), including the rossmann fold with the acceptor-binding domain and the stem-contact fold. this glurs-like protein, which lacks the anticodon-binding domain, does not use trna(glu) as substrate in vitro nor in vivo, but aminoacylates trna(asp) with glutamate. the yadb gene is expressed in wild-type e. coli as an operon with the dksa gene, which encodes a protein involv ... | 2004 | 15096594 |
| a truncated aminoacyl-trna synthetase modifies rna. | aminoacyl-trna synthetases are modular enzymes composed of a central active site domain to which additional functional domains were appended in the course of evolution. analysis of bacterial genome sequences revealed the presence of many shorter aminoacyl-trna synthetase paralogs. here we report the characterization of a well conserved glutamyl-trna synthetase (glurs) paralog (yadb in escherichia coli) that is present in the genomes of >40 species of proteobacteria, cyanobacteria, and actinobact ... | 2004 | 15096612 |
| specific recognition of rpso mrna and 16s rrna by escherichia coli ribosomal protein s15 relies on both mimicry and site differentiation. | the ribosomal protein s15 binds to 16s rrna, during ribosome assembly, and to its own mrna (rpso mrna), affecting autocontrol of its expression. in both cases, the rna binding site is bipartite with a common subsite consisting of a g*u/g-c motif. the second subsite is located in a three-way junction in 16s rrna and in the distal part of a stem forming a pseudoknot in escherichia coli rpso mrna. to determine the extent of mimicry between these two rna targets, we determined which amino acids inte ... | 2004 | 15101974 |
| crystal structures of escherichia coli topoisomerase iv pare subunit (24 and 43 kilodaltons): a single residue dictates differences in novobiocin potency against topoisomerase iv and dna gyrase. | topoisomerase iv and dna gyrase are related bacterial type ii topoisomerases that utilize the free energy from atp hydrolysis to catalyze topological changes in the bacterial genome. the essential function of dna gyrase is the introduction of negative dna supercoils into the genome, whereas the essential function of topoisomerase iv is to decatenate daughter chromosomes following replication. here, we report the crystal structures of a 43-kda n-terminal fragment of escherichia coli topoisomerase ... | 2004 | 15105144 |
| thermal and conformational stability of ssh10b protein from archaeon sulfolobus shibattae. | the secondary structure of the dna binding protein ssh10b is largely unaffected by change in temperature between 25 degrees c and 85 degrees c, indicating that the protein is highly thermostable. here, we report the temperature-dependent equilibrium denaturation of ssh10b in the presence of guanidine hydrochloride (gdnhcl). it was found that the transition midpoint values of the temperature (t(m)), and changes of enthalpy (deltah(m)) and entropy (deltas(m)) of ssh10b unfolding were linearly decr ... | 2004 | 15107015 |
| ring-shaped architecture of recr: implications for its role in homologous recombinational dna repair. | recr, together with recf and reco, facilitates reca loading in the recf pathway of homologous recombinational dna repair in procaryotes. the human rad52 protein is a functional counterpart of recfor. we present here the crystal structure of recr from deinococcus radiodurans (dr recr). a monomer of dr recr has a two-domain structure: the n-terminal domain with a helix-hairpin-helix (hhh) motif and the c-terminal domain with a cys4 zinc-finger motif, a toprim domain and a walker b motif. four such ... | 2004 | 15116069 |
| distinctive protein signatures provide molecular markers and evidence for the monophyletic nature of the deinococcus-thermus phylum. | the deinococcus-thermus group of species is currently recognized as a distinct phylum solely on the basis of their branching in 16s rrna trees. no unique biochemical or molecular characteristics that can distinguish this group from all other bacteria are known at present. in this work, we describe eight conserved indels (viz., inserts or deletions) in seven widely distributed proteins that are distinctive characteristics of the deinococcus-thermus phylum but are not found in any other group of b ... | 2004 | 15126471 |
| comparison of two real-time quantitative assays for detection of severe acute respiratory syndrome coronavirus. | the new severe acute respiratory syndrome (sars) coronavirus (cov), described in february 2003, infected a total of 8,439 people. a total of 812 people died due to respiratory insufficiency. close contact with symptomatic patients appeared to be the main route of transmission. however, potential transmission by blood transfusion could not be definitely excluded. two real-time sars-specific pcr assays were assessed for their sensitivities, agreement of test results, and intra-assay variabilities. ... | 2004 | 15131175 |
| thermus thermophilus genome analysis: benefits and implications. | the genome sequence analysis of thermus thermophilus hb27, a microorganism with high biotechnological potential, has recently been published. in that report, the chromosomal and the megaplasmid sequence were compared to those of other organisms and discussed on the basis of their physiological and metabolic features. out of the 2,218 putative genes identified through the large genome sequencing project, a significant number has potential interest for biotechnology. the present communication will ... | 2004 | 15134584 |
| mycobacterium tuberculosis dnaa initiator protein: purification and dna-binding requirements. | the mycobacterium tuberculosis oric (the origin of chromosomal replication) region contains 13 non-perfect dnaa boxes. the m. tuberculosis initiator protein, dnaa, was overexpressed in escherichia coli as a soluble his-tagged fusion protein. the purified protein his6mtdnaa was investigated for its binding properties to dnaa boxes from the oric region. gel retardation demonstrated that the dnaa from m. tuberculosis requires two dnaa boxes for efficient binding. electron microscopy as well as dnas ... | 2004 | 15137907 |
| resistance of rumen bacteria murein to bovine gastric lysozyme. | lysozymes, enzymes mostly associated with defence against bacterial infections, are mureinolytic. ruminants have evolved a gastric c type lysozyme as a digestive enzyme, and profit from digestion of foregut bacteria, after most dietary components, including protein, have been fermented in the rumen. in this work we characterized the biological activities of bovine gastric secretions against membranes, purified murein and bacteria. | 2004 | 15137912 |
| mapping of the second tetracycline binding site on the ribosomal small subunit of e.coli. | tetracycline blocks stable binding of aminoacyl-trna to the bacterial ribosomal a-site. various tetracycline binding sites have been identified in crystals of the 30s ribosomal small subunit of thermus thermophilus. here we describe a direct photo- affinity modification of the ribosomal small subunits of escherichia coli with 7-[3h]-tetracycline. to select for specific interactions, an excess of the 30s subunits over tetracycline has been used. primer extension analysis of the 16s rrna revealed ... | 2004 | 15141029 |
| the structure of a ribosomal protein s8/spc operon mrna complex. | in bacteria, translation of all the ribosomal protein cistrons in the spc operon mrna is repressed by the binding of the product of one of them, s8, to an internal sequence at the 5' end of the l5 cistron. the way in which the first two genes of the spc operon are regulated, retroregulation, is mechanistically distinct from translational repression by s8 of the genes from l5 onward. a 2.8 a resolution crystal structure has been obtained of escherichia coli s8 bound to this site. despite sequence ... | 2004 | 15146079 |
| a minimalist glutamyl-trna synthetase dedicated to aminoacylation of the trnaasp quc anticodon. | escherichia coli encodes yadb, a protein displaying 34% identity with the catalytic core of glutamyl-trna synthetase but lacking the anticodon-binding domain. we show that yadb is a trna modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the trna(asp) quc anticodon. this conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate trna(asp) isolated from an e.coli trna-guanosine transgl ... | 2004 | 15150343 |
| simulation, experiment, and evolution: understanding nucleation in protein s6 folding. | in this study, we explore nucleation and the transition state ensemble of the ribosomal protein s6 using a monte carlo (mc) go model in conjunction with restraints from experiment. the results are analyzed in the context of extensive experimental and evolutionary data. the roles of individual residues in the folding nucleus are identified, and the order of events in the s6 folding mechanism is explored in detail. interpretation of our results agrees with, and extends the utility of, experiments ... | 2004 | 15150413 |
| new enzymes from environmental cassette arrays: functional attributes of a phosphotransferase and an rna-methyltransferase. | by targeting gene cassettes by polymerase chain reaction (pcr) directly from environmentally derived dna, we are able to amplify entire open reading frames (orfs) independently of prior sequence knowledge. approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. here we describe the characterization of two orfs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with aph(7") f ... | 2004 | 15152095 |
| crystal structure of escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/atp-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets. | cytidine triphosphate synthetases (ctpss) produce ctp from utp and glutamine, and regulate intracellular ctp levels through interactions with the four ribonucleotide triphosphates. we solved the 2.3-a resolution crystal structure of escherichia coli ctps using hg-mad phasing. the structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase n-terminal domain and a type 1 glutamine amidotransferase c-terminal domain. for ... | 2004 | 15157079 |
| crystal structure of the deinococcus radiodurans single-stranded dna-binding protein suggests a mechanism for coping with dna damage. | single-stranded dna (ssdna)-binding (ssb) proteins are uniformly required to bind and protect single-stranded intermediates in dna metabolic pathways. all bacterial and eukaryotic ssb proteins studied to date oligomerize to assemble four copies of a conserved domain, called an oligonucleotide/oligosaccharide-binding (ob) fold, that cooperate in nonspecific ssdna binding. the vast majority of bacterial ssb family members function as homotetramers, with each monomer contributing a single ob fold. ... | 2004 | 15159541 |
| the ribosomal protein rps15p is required for nuclear exit of the 40s subunit precursors in yeast. | we have conducted a genetic screen in order to identify ribosomal proteins of saccharomyces cerevisiae involved in nuclear export of the small subunit precursors. this has led us to distinguish rps15p as a protein dispensable for maturation of the pre-40s particles, but whose assembly into the pre-ribosomes is a prerequisite to their nuclear exit. upon depletion of rps15p, 20s pre-rrna is released from the nucleolus and retained in the nucleus, without alteration of the pre-rrna early cleavages. ... | 2004 | 15167894 |
| identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases. | a combination of sequence homology analyses of mevalonate diphosphate decarboxylase (mdd) proteins and structural information for mdd leads to the hypothesis that asp 302 and lys 18 are active site residues in mdd. these residues were mutated to replace acidic/basic side chains and the mutant proteins were isolated and characterized. binding and competitive displacement studies using trinitrophenyl-atp, a fluorescent analog of substrate atp, indicate that these mutant enzymes (d302a, d302n, k18m ... | 2004 | 15169949 |
| visualization of ribosome-recycling factor on the escherichia coli 70s ribosome: functional implications. | after the termination step of protein synthesis, a deacylated trna and mrna remain associated with the ribosome. the ribosome-recycling factor (rrf), together with elongation factor g (ef-g), disassembles this posttermination complex into mrna, trna, and the ribosome. we have obtained a three-dimensional cryo-electron microscopic map of a complex of the escherichia coli 70s ribosome and rrf. we find that rrf interacts mainly with the segments of the large ribosomal subunit's (50s) rrna helices t ... | 2004 | 15178758 |
| cloning, sequencing, and characterization of a heat- and alkali-stable type i pullulanase from anaerobranca gottschalkii. | the gene encoding a type i pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium anaerobranca gottschalkii. in addition, the homologous gene was isolated from a gene library of anaerobranca horikoshii and sequenced. the proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria fervidobacterium pennivorans and thermotoga maritima. the pullulanase gene from a. gottschalkii (en ... | 2004 | 15184138 |
| interaction of horse heart and thermus thermophilus type c cytochromes with phospholipid vesicles and hydrophobic surfaces. | the binding of horse heart cytochrome c (cyt-c) and thermus thermophilus cytochrome c(552) (cyt-c(552)) to dioleoyl phosphatidylglycerol (dopg) vesicles was investigated using fourier transform infrared (ftir) spectroscopy and turbidity measurements. ftir spectra revealed that the tertiary structures of both cytochromes became more open when bound to dopg vesicles, but this was more pronounced for cyt-c. their secondary structures were unchanged. turbidity measurements showed important differenc ... | 2004 | 15189883 |
| interactions between uvra and uvrb: the role of uvrb's domain 2 in nucleotide excision repair. | nucleotide excision repair (ner) is a highly conserved dna repair mechanism present in all kingdoms of life. uvrb is a central component of the bacterial ner system, participating in damage recognition, strand excision and repair synthesis. none of the three presently available crystal structures of uvrb has defined the structure of domain 2, which is critical for the interaction with uvra. we have solved the crystal structure of the uvrb y96a variant, which reveals a new fold for domain 2 and i ... | 2004 | 15192705 |
| thiostrepton-resistant mutants of thermus thermophilus. | ribosomal protein l11 and its associated binding site on 23s rrna together comprise one of the principle components that mediate interactions of translation factors with the ribosome. this site is also the target of the antibiotic thiostrepton, which has been proposed to act by preventing important structural transitions that occur in this region of the ribosome during protein synthesis. here, we describe the isolation and characterization of spontaneous thiostrepton-resistant mutants of the ext ... | 2004 | 15199170 |
| the role of bacterial antizyme: from an inhibitory protein to atoc transcriptional regulator. | this review considers the role of bacterial antizyme in the regulation of polyamine biosynthesis and gives new perspectives on the involvement of antizyme in other significant cellular mechanisms. antizyme is a protein molecule induced by the end product of the enzymic reaction that it inhibits, in a non-competitive manner. the bacterial ornithine decarboxylase is regulated by nucleotides, phosphorylation and antizyme. the inhibition of ornithine decarboxylase by antizyme can be relieved to diff ... | 2004 | 15200682 |
| a gene from the mesophilic bacterium dehalococcoides ethenogenes encodes a novel mannosylglycerate synthase. | mannosylglycerate (mg) is a common compatible solute found in thermophilic and hyperthermophilic prokaryotes. in this study we characterized a mesophilic and bifunctional mannosylglycerate synthase (mgsd) encoded in the genome of the bacterium dehalococcoides ethenogenes. mgsd encodes two domains with extensive homology to mannosyl-3-phosphoglycerate synthase (mpgs, ec 2.4.1.217) and to mannosyl-3-phosphoglycerate phosphatase (mpgp, ec 3.1.3.70), which catalyze the consecutive synthesis and deph ... | 2004 | 15205409 |
| two distinct domains of the beta subunit of aquifex aeolicus leucyl-trna synthetase are involved in trna binding as revealed by a three-hybrid selection. | the aquifex aeolicus alphabeta-leurs is the only known heterodimeric class ia aminoacyl-trna synthetase. in this study, we investigated the function of the beta subunit which is believed to bind trna(leu). a yeast three-hybrid system was constructed on the basis of the interaction of the beta subunit with its cognate trna(leu). then, seven mutated beta subunits exhibiting impaired trna binding capacities were selected out from a randomly mutated library. two mutations were identified in the clas ... | 2004 | 15208367 |
| direct evidence that a conserved arginine in ruvb aaa+ atpase acts as an allosteric effector for the atpase activity of the adjacent subunit in a hexamer. | the escherichia coli ruva and ruvb protein complex promotes branch migration of holliday junctions during recombinational repair and homologous recombination and at stalled replication forks. the ruvb protein belongs to the aaa(+) (atpase associated with various cellular activities) atpase family and forms a hexameric ring in an atp-dependent manner. studies on the oligomeric aaa(+) class atpases suggest that a conserved arginine residue is located in close proximity to the atpase site of the ad ... | 2004 | 15210950 |
| crystal structure of elongation factor p from thermus thermophilus hb8. | translation elongation factor p (ef-p) stimulates ribosomal peptidyltransferase activity. ef-p is conserved in bacteria and is essential for cell viability. eukarya and archaea have an ef-p homologue, eukaryotic initiation factor 5a (eif-5a). in the present study, we determined the crystal structure of ef-p from thermus thermophilus hb8 at a 1.65-a resolution. ef-p consists of three beta-barrel domains (i, ii, and iii), whereas eif-5a has only two domains (n and c domains). domain i of ef-p is t ... | 2004 | 15210970 |
| molecular characterization of benzimidazole resistance in helicobacter pylori. | a family of benzimidazole derivatives (bi) was shown to possess potent and selective activity against helicobacter pylori, although the precise cellular target of the bis is unknown. spontaneous h. pylori mutants were isolated as resistant to a representative bi (compound a). genomic dna was isolated from a bi-resistant mutant, transformed into a bi-sensitive strain, and found to be sufficient to confer bi resistance. the resistance determinant was localized to a 17-kb clone after screening a la ... | 2004 | 15215104 |
| the crystal structure of human endonuclease viii-like 1 (neil1) reveals a zincless finger motif required for glycosylase activity. | in prokaryotes, two dna glycosylases recognize and excise oxidized pyrimidines: endonuclease iii (nth) and endonuclease viii (nei). the oxidized purine 8-oxoguanine, on the other hand, is recognized by fpg (also known as mutm), a glycosylase that belongs to the same family as nei. the recent availability of the human genome sequence allowed the identification of three human homologs of escherichia coli nei. we report here the crystal structure of a human nei-like (neil) enzyme, neil1. the struct ... | 2004 | 15232006 |
| thermus thermophilus as a cell factory for the production of a thermophilic mn-dependent catalase which fails to be synthesized in an active form in escherichia coli. | thermostable mn-dependent catalases are promising enzymes in biotechnological applications as h(2)o(2)-detoxifying systems. we cloned the genes encoding mn-dependent catalases from thermus thermophilus hb27 and hb8 and a less thermostable mutant carrying two amino acid replacements (m129v and e293g). when the wild-type and mutant genes were overexpressed in escherichia coli, unmodified or six-his-tagged proteins of the expected size were overproduced as inactive proteins. several attempts to obt ... | 2004 | 15240253 |
| identification of a bifunctional enzyme mnmc involved in the biosynthesis of a hypermodified uridine in the wobble position of trna. | the gene encoding the bifunctional enzyme mnmc that catalyzes the two last steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm5s2u) in trna has been previously mapped at about 50 min on the escherichia coli k12 chromosome, but to date the identity of the corresponding enzyme has not been correlated with any of the known open reading frames (orfs). using the protein fold-recognition approach, we predicted that the 74-kda product of the yfck orf located at 52.6 min and annotated as ... | 2004 | 15247431 |
| the phosphoenolpyruvate carboxylase from methanothermobacter thermautotrophicus has a novel structure. | in methanothermobacter thermautotrophicus, oxaloacetate synthesis is a major and essential co(2)-fixation reaction. this methanogenic archaeon possesses two oxaloacetate-synthesizing enzymes, pyruvate carboxylase and phosphoenolpyruvate carboxylase. the phosphoenolpyruvate carboxylase from this organism was purified to homogeneity. the subunit size of this homotetrameric protein was 55 kda, which is about half that of all known bacterial and eukaryotic phosphoenolpyruvate carboxylases (ppcs). th ... | 2004 | 15262949 |
| discrimination against deoxyribonucleotide substrates by bacterial rna polymerase. | nucleic acid polymerases have evolved elaborate mechanisms that prevent incorporation of the non-cognate substrates, which are distinguished by both the base and the sugar moieties. while the mechanisms of substrate selection have been studied in single-subunit dna and rna polymerases (dnaps and rnaps, respectively), the determinants of substrate binding in the multisubunit rnaps are not yet known. molecular modeling of thermus thermophilus rnap-substrate ntp complex identified a conserved beta' ... | 2004 | 15262972 |
| heteronuclear nmr investigations of dynamic regions of intact escherichia coli ribosomes. | 15n-(1)h nmr spectroscopy has been used to probe the dynamic properties of uniformly (15)n labeled escherichia coli ribosomes. despite the high molecular weight of the complex ( approximately 2.3 mda), [(1)h-(15)n] heteronuclear single-quantum correlation spectra contain approximately 100 well resolved resonances, the majority of which arise from two of the four c-terminal domains of the stalk proteins, l7/l12. heteronuclear pulse-field gradient nmr experiments show that the resonances arise fro ... | 2004 | 15263071 |
| formation and characterization of an all-ferrous rieske cluster and stabilization of the [2fe-2s]0 core by protonation. | the all-ferrous rieske cluster, [2fe-2s](0), has been produced in solution and characterized by protein-film voltammetry and uv-visible, epr, and mössbauer spectroscopies. the [2fe-2s](0) cluster, in the overexpressed soluble domain of the rieske protein from the bovine cytochrome bc(1) complex, is formed at -0.73 v at ph 7. therefore, at ph 7, the [2fe-2s](1+/0) couple is 1.0 v below the [2fe-2s](2+/1+) couple. the two cluster-bound ferrous irons are both high spin (s = 2), and they are coupled ... | 2004 | 15263097 |
| insights into the dna repair process by the formamidopyrimidine-dna glycosylase investigated by molecular dynamics. | formamidopyrimidine-dna glycosylase (fpg) identifies and removes 8-oxoguanine from dna. all of the x-ray structures of fpg complexed to an abasic site containing dna exhibit a common disordered region present in the c-terminal domain of the enzyme. however, this region is believed to be involved in the damaged base binding site when the initial protein/dna complex is formed. the dynamic behavior of the disordered polypeptide (named loop) in relation to the supposed scenario for the dna repair me ... | 2004 | 15273302 |
| staphylococcus aureus dna ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay. | dna ligases are key enzymes involved in the repair and replication of dna. prokaryotic dna ligases uniquely use nad+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use atp. this difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. we have developed a homogeneous chemiluminescence-based hybridization protection assay for staphylococcus aureus dna ligase that uses novel acridinium ester technology and demonstrate that ... | 2004 | 15283677 |
| a single-amino-acid lid renders a gas-tight compartment within a membrane-bound transporter. | proteins undergo structural fluctuations between nearly isoenergetic substates. such fluctuations are often intimately linked with the functional properties of proteins. however, in some cases, such as in transmembrane ion transporters, the control of the ion transport requires that the protein is designed to restrict the motions in specific regions. in this study, we have investigated the dynamics of a membrane-bound respiratory oxidase, which acts both as an enzyme catalyzing reduction of o(2) ... | 2004 | 15289603 |
| targeting the a site rna of the escherichia coli ribosomal 30 s subunit by 2'-o-methyl oligoribonucleotides: a quantitative equilibrium dialysis binding assay and differential effects of aminoglycoside antibiotics. | the bacterial ribosome comprises 30 s and 50 s ribonucleoprotein subunits, contains a number of binding sites for known antibiotics and is an attractive target for selection of novel antibacterial agents. on the 30 s subunit, for example, the a site (aminoacyl site) close to the 3'-end of 16 s rrna is highly important in the decoding process. binding by some aminoglycoside antibiotics to the a site leads to erroneous protein synthesis and is lethal for bacteria. we targeted the a site on purifie ... | 2004 | 15294017 |
| construction of a chimeric thermostable pyrophosphatase to facilitate its purification and immobilization by using the choline-binding tag. | the thermophilic inorganic pyrophosphatase (pyr) from thermus thermophilus has been produced in escherichia coli fused to the c terminus of the choline-binding tag (chb tag) derived from the choline-binding domain (chbd) of pneumococcal lyta autolysin. the chimeric chbd-pyr protein retains its thermostable activity and can be purified in a single step by deae-cellulose affinity chromatography. pyr can be further released from the chbd by thrombin, using the specific protease recognition site inc ... | 2004 | 15294797 |
| stabilizing roles of residual structure in the empty heme binding pockets and unfolded states of microsomal and mitochondrial apocytochrome b5. | the microsomal (mc) and mitochondrial (om) isoforms of mammalian cytochrome b5 are the products of different genes, which likely arose via duplication of a primordial gene and subsequent functional divergence. despite sharing essentially identical folds, heme-polypeptide interactions are stronger in om b5s than in mc b5s due to the presence of two conserved patches of hydrophobic amino acid side chains in the om heme binding pockets. this is of fundamental interest in terms of understanding heme ... | 2004 | 15295112 |
| creating ribosomes with an all-rna 30s subunit p site. | ribosome crystal structures have revealed that two small subunit proteins, s9 and s13, have c-terminal tails, which, together with several features of 16s rrna, contact the anticodon stem-loop of p-site trna. to test the functional importance of these protein tails, we created genomic deletions of the c-terminal regions of s9 and s13. all of the tail deletions, including double mutants containing deletions in both s9 and s13, were viable, showing that escherichia coli cells can synthesize all of ... | 2004 | 15308780 |
| thermus thermophilus l11 methyltransferase, prma, is dispensable for growth and preferentially modifies free ribosomal protein l11 prior to ribosome assembly. | the ribosomal protein l11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the l11 methyltransferase prma, the product of the prma gene. the role of l11 methylation in ribosome function or assembly has yet to be determined, although the deletion of escherichia coli prma has no apparent phenotype. we have constructed a mutant of the extreme thermophile thermus thermophilus in which the prma gene has been disrupted with the htk gene encoding a heat-stable kanamy ... | 2004 | 15317787 |
| substrate-induced asymmetry and channel closure revealed by the apoenzyme structure of mycobacterium tuberculosis phosphopantetheine adenylyltransferase. | phosphopantetheine adenylyltransferase (ppat) catalyzes the penultimate step in prokaryotic coenzyme a (coa) biosynthesis, directing the transfer of an adenylyl group from atp to 4'-phosphopantetheine (ppant) to yield dephospho-coa (dpcoa). the crystal structures of escherichia coli ppat bound to its substrates, product, and inhibitor revealed an allosteric hexameric enzyme with half-of-sites reactivity, and established an in-line displacement catalytic mechanism. to provide insight into the mec ... | 2004 | 15322293 |
| incidence of antibiotic resistance in campylobacter jejuni isolated in alberta, canada, from 1999 to 2002, with special reference to tet(o)-mediated tetracycline resistance. | of 203 human clinical isolates of campylobacter jejuni from alberta, canada (1999 to 2002), 101 isolates (50%) were resistant to at least 64 microg of tetracycline/ml, with four isolates exhibiting higher levels of tetracycline resistance (512 microg/ml). in total, the mics for 37% of tetracycline-resistant isolates (256 to 512 microg/ml) were higher than those previously reported in c. jejuni (64 to 128 microg/ml). in the tetracycline-resistant clinical isolates, 67% contained plasmids and all ... | 2004 | 15328109 |
| dna ligases ensure fidelity by interrogating minor groove contacts. | dna ligases, found in both prokaryotes and eukaryotes, covalently link the 3'-hydroxyl and 5'-phosphate ends of duplex dna segments. this reaction represents a completion step for dna replication, repair and recombination. it is well established that ligases are sensitive to mispairs present on the 3' side of the ligase junction, but tolerant of mispairs on the 5' side. while such discrimination would increase the overall accuracy of dna replication and repair, the mechanisms by which this fidel ... | 2004 | 15328364 |
| photolabile anticodon stem-loop analogs of trnaphe as probes of ribosomal structure and structural fluctuation at the decoding center. | with the recent availability of high-resolution structures of bacterial ribosomes, studies of ribosome-catalyzed protein biosynthesis are now focusing on the nature of conformational changes that occur as the ribosome exerts its complex catalytic function. photocrosslinking can be relevant for this purpose by providing clues to ribosomal structural fluctuations and dynamics. here we describe crosslinking experiments on 70s ribosomes using two photolabile anticodon stem-loop derivatives of escher ... | 2004 | 15337844 |
| crystal structure of ribosomal protein l27 from thermus thermophilus hb8. | ribosomal protein l27 is located near the peptidyltransferase center at the interface of ribosomal subunits, and is important for ribosomal assembly and function. we report the crystal structure of ribosomal protein l27 from thermus thermophilus hb8, which was determined by the multiwavelength anomalous dispersion method and refined to an r-factor of 19.7% (r(free) = 23.6%) at 2.8 a resolution. the overall fold is an all beta-sheet hybrid. it consists of two sets of four-stranded beta-sheets for ... | 2004 | 15340170 |
| 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 ... | 2003 | 14984054 |
| 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 ... | 2003 | 12505993 |
| 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 ... | 2003 | 12513990 |
| uptake and antifungal activity of oligonucleotides in candida albicans. | candida albicans is a significant cause of disease in immunocompromised humans. because the number of people infected by fungal pathogens is increasing, strategies are being developed to target rnas in fungi. this work shows that oligonucleotides can serve as therapeutics against c. albicans. in particular, oligonucleotides are taken up from cell culture medium in an energy-dependent process. after uptake, oligonucleotides, including rna, remain mostly intact after 12 h in culture. for culture c ... | 2003 | 12552085 |
| atp binding by glutamyl-trna synthetase is switched to the productive mode by trna binding. | aminoacyl-trna synthetases catalyze the formation of an aminoacyl-amp from an amino acid and atp, prior to the aminoacyl transfer to trna. a subset of aminoacyl-trna synthetases, including glutamyl-trna synthetase (glurs), have a regulation mechanism to avoid aminoacyl-amp formation in the absence of trna. in this study, we determined the crystal structure of the 'non-productive' complex of thermus thermophilus glurs, atp and l-glutamate, together with those of the glurs.atp, glurs.trna.atp and ... | 2003 | 12554668 |
| the crystal structure of a 26-nucleotide rna containing a hook-turn. | a crystal structure has been obtained for a 26-nucleotide rna that contains the loop e sequence from chromatium minutissimum. rather than having a loop e-like conformation, it consists of an a-form helix that splits into two separate strands following a sheared a-g base pair. the backbone of the strand containing the g of the a-g pair makes a turn of almost 180 degrees in the space of two nucleotides, and then interacts with the minor groove of the helix from which it originates. similar structu ... | 2003 | 12554875 |
| mechanism of molecular interactions for trna(val) recognition by valyl-trna synthetase. | the molecular interactions between valyl-trna synthetase (valrs) and trna(val), with the c34-a35-c36 anticodon, from thermus thermophilus were studied by crystallographic analysis and structure-based mutagenesis. in the valrs-bound structure of trna(val), the successive a35-c36 residues (the major identity elements) of trna(val) are base-stacked upon each other, and fit into a pocket on the alpha-helix bundle domain of valrs. hydrogen bonds are formed between valrs and a35-c36 of trna(val) in a ... | 2003 | 12554880 |
| the deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by ispg and isph protein. | earlier in vivo studies have shown that the sequential action of the ispg and isph proteins is essential for the reductive transformation of 2c-methyl-d-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(e)-butenyl 4-diphosphate. a recombinant fusion protein comprising maltose binding protein and ispg protein domains was purified from a recombinant escherichia coli strain. the purified protein failed to transform 2c-methyl-d-eryth ... | 2003 | 12571359 |
| the structural basis of cysteine aminoacylation of trnapro by prolyl-trna synthetases. | cysteinyl-trna synthetase is an essential enzyme required for protein synthesis. genes encoding this protein have not been identified in methanocaldococcus jannaschii, methanothermobacter thermautotrophicus, or methanopyrus kandleri. it has previously been proposed that the prolyl-trna synthetase (prors) enzymes in these organisms recognize either proline or cysteine and can aminoacylate their cognate trnas through a dual-specificity mechanism. we report five crystal structures at resolutions be ... | 2003 | 12578991 |
| evidence for rotation of v1-atpase. | v(o)v(1)-atpase is responsible for acidification of eukaryotic intracellular compartments and atp synthesis of archaea and some eubacteria. from the similarity to f(o)f(1)-atp synthase, v(o)v(1)-atpase has been assumed to be a rotary motor, but to date there are no experimental data to support this. here we visualized the rotation of single molecules of v(1)-atpase, a catalytic subcomplex of v(o)v(1)-atpase. v(1)-atpase from thermus thermophilus was immobilized onto a glass surface, and a bead w ... | 2003 | 12598655 |
| orientation and interactions of an essential tryptophan (trp-38) in the capsid subunit of pf3 filamentous virus. | the filamentous bacteriophage pf3 consists of a covalently closed dna single strand of 5833 nucleotides sheathed by approximately 2500 copies of a 44-residue capsid subunit. the capsid subunit contains a single tryptophan residue (trp-38), which is located within the basic c-terminal sequence (-rwikaqff) and is essential for virion assembly in vivo. polarized raman microspectroscopy has been employed to determine the orientation of the trp-38 side chain in the native virus structure. the polariz ... | 2003 | 12609899 |
| genetic diversity: frameshift mechanisms alter coding of a gene (epstein-barr virus lf3 gene) that contains multiple 102-base-pair direct sequence repeats. | frameshift mutations provide recognized mechanisms for changing the coding potential of an organism. here, multiple frameshifts are identified in repetitive sequences within an epstein-barr virus unspliced early gene, lf3, which is associated with the viral replicative cycle and also transcriptionally expressed in many virally associated tumors. on the dna strand encoding lf3, there are three open reading frames, only one of which contains an initiation codon. most (>95%) of the gene consists of ... | 2003 | 12612089 |
| the feci extracytoplasmic-function sigma factor of escherichia coli interacts with the beta' subunit of rna polymerase. | transcription of the ferric citrate transport system of escherichia coli k-12 is mediated by the extracytoplasmic-function (ecf) sigma factor feci, which is activated by ferric citrate in the growth medium. by using a bacterial two-hybrid system, it was shown in vivo that feci binds to the beta' subunit of rna polymerase. the inactive mutant protein feci(k155e) displayed reduced binding to beta', and small deletions along the entire feci protein led to total impairment of beta' binding. in vitro ... | 2003 | 12618442 |
| the rhomboids: a nearly ubiquitous family of intramembrane serine proteases that probably evolved by multiple ancient horizontal gene transfers. | the rhomboid family of polytopic membrane proteins shows a level of evolutionary conservation unique among membrane proteins. they are present in nearly all the sequenced genomes of archaea, bacteria and eukaryotes, with the exception of several species with small genomes. on the basis of experimental studies with the developmental regulator rhomboid from drosophila and the aara protein from the bacterium providencia stuartii, the rhomboids are thought to be intramembrane serine proteases whose ... | 2003 | 12620104 |
| in situ accessibility of small-subunit rrna of members of the domains bacteria, archaea, and eucarya to cy3-labeled oligonucleotide probes. | low accessibility of the rrna is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes. in this study we compare accessibility data for the 16s rrna of escherichia coli (gamma proteobacteria, bacteria) with the phylogenetically distantly related organisms pirellula sp. strain 1 (planctomycetes, bacteria) and metallosphaera sedula (crenarchaeota, archaea) and ... | 2003 | 12620867 |
| characterization of a novel thermostable o-acetylserine sulfhydrylase from aeropyrum pernix k1. | an o-acetylserine sulfhydrylase (oass) from the hyperthermophilic archaeon aeropyrum pernix k1, which shares the pyridoxal 5'-phosphate binding motif with both oass and cystathionine beta-synthase (cbs), was cloned and expressed by using escherichia coli rosetta(de3). the purified protein was a dimer and contained pyridoxal 5'-phosphate. it was shown to be an enzyme with cbs activity as well as oass activity in vitro. the enzyme retained 90% of its activity after a 6-h incubation at 100 degrees ... | 2003 | 12644499 |
| antagonistic signals within the cox2 mrna coding sequence control its translation in saccharomyces cerevisiae mitochondria. | translation of the mitochondrially coded cox2 mrna within the organelle in yeast produces the precursor of cox2p (pre-cox2p), which is processed and assembled into cytochrome c oxidase. the mrna sequence of the first 14 cox2 codons, specifying the pre-cox2p leader peptide, was previously shown to contain a positively acting element required for translation of a mitochondrial reporter gene, arg8(m), fused to the 91st codon of cox2. here we show that three relatively short sequences within the cox ... | 2003 | 12649494 |
| the yeast eif3 subunits tif32/a, nip1/c, and eif5 make critical connections with the 40s ribosome in vivo. | initiation factor 3 (eif3) forms a multifactor complex (mfc) with eif1, eif2, and eif5 that stimulates met-trna(i)(met) binding to 40s ribosomes and promotes scanning or aug recognition. we have previously characterized mfc subcomplexes produced in vivo from affinity-tagged eif3 subunits lacking discrete binding domains for other mfc components. here we asked whether these subcomplexes can bind to 40s ribosomes in vivo. we found that the n- and c-terminal domains of nip1/eif3c, the n- and c-term ... | 2003 | 12651896 |
| high-efficiency generation of antibiotic-resistant strains of streptococcus pneumoniae by pcr and transformation. | we designed a method by which to generate antibiotic-resistant strains of streptococcus pneumoniae at frequencies 4 orders of magnitude greater than the spontaneous mutation rate. the method is based on the natural ability of this organism to be genetically transformed with pcr products carrying sequences homologous to its chromosome. the genes encoding the targets of ciprofloxacin (parc, encoding the parc subunit of dna topoisomerase iv), rifampin (rpob, encoding the beta subunit of rna polymer ... | 2003 | 12654655 |
| non-discriminating and discriminating aspartyl-trna synthetases differ in the anticodon-binding domain. | in most organisms, trna aminoacylation is ensured by 20 aminoacyl-trna synthetases (aarss). in eubacteria, however, synthetases can be duplicated as in thermus thermophilus, which contains two distinct asprss. while asprs-1 is specific, asprs-2 is non-discriminating and aspartylates trna(asp) and trna(asn). the structure at 2.3 a resolution of asprs-2, the first of a non-discriminating synthetase, was solved. it differs from that of asprs-1 but has resemblance to that of discriminating and archa ... | 2003 | 12660169 |
| phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core. | the examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. we developed a method for aseptically sampling a sediment-containing portion of a greenland ice core that had remained at -9 degrees c for over 100,000 years. epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. anaerobic enrichment cultures inoculated w ... | 2003 | 12676695 |
| ribosomal protein s15 represses its own translation via adaptation of an rrna-like fold within its mrna. | the 16s rrna-binding ribosomal protein s15 is a key component in the assembly of the small ribosomal subunit in bacteria. we have shown that s15 from the extreme thermophile thermus thermophilus represses the translation of its own mrna in vitro, by interacting with the leader segment of its mrna. the s15 mrna-binding site was characterized by footprinting experiments, deletion analysis and site-directed mutagenesis. s15 binding triggers a conformational rearrangement of its mrna into a fold tha ... | 2003 | 12682022 |
| a novel uracil-dna glycosylase family related to the helix-hairpin-helix dna glycosylase superfamily. | cytosine bases can be deaminated spontaneously to uracil, causing dna damage. uracil-dna glycosylase (udg), a ubiquitous uracil-excising enzyme found in bacteria and eukaryotes, is one of the enzymes that repair this kind of dna damage. to date, no udg-coding gene has been identified in methanococcus jannaschii, although its entire genome was deciphered. here, we have identified and characterized a novel udg from m.jannaschii designated as mjudg. it efficiently removed uracil from both single- a ... | 2003 | 12682355 |
| cloning and characterization of trna (m1a58) methyltransferase (trmi) from thermus thermophilus hb27, a protein required for cell growth at extreme temperatures. | n1-methyladenosine (m1a) is found at position 58 in the t-loop of many trnas. in yeast, the formation of this modified nucleoside is catalyzed by the essential trna (m1a58) methyltransferase, a tetrameric enzyme that is composed of two types of subunits (gcd14p and gcd10p). in this report we describe the cloning, expression and characterization of a gcd14p homolog from the hyperthermophilic bacterium thermus thermophilus. the purified recombinant enzyme behaves as a homotetramer of 150 kda by ge ... | 2003 | 12682365 |
| glycyl trna synthetase mutations in charcot-marie-tooth disease type 2d and distal spinal muscular atrophy type v. | charcot-marie-tooth disease type 2d (cmt2d) and distal spinal muscular atrophy type v (dsma-v) are axonal peripheral neuropathies inherited in an autosomal dominant fashion. our previous genetic and physical mapping efforts localized the responsible gene(s) to a well-defined region on human chromosome 7p. here, we report the identification of four disease-associated missense mutations in the glycyl trna synthetase gene in families with cmt2d and dsma-v. this is the first example of an aminoacyl ... | 2003 | 12690580 |
| structure-function studies of escherichia coli rpoh (sigma32) by in vitro linker insertion mutagenesis. | the sigma factor rpoh (sigma(32)) is the key regulator of the heat shock response in escherichia coli. many structural and functional properties of the sigma factor are poorly understood. to gain further insight into rpoh regions that are either important or dispensable for its cellular activity, we generated a collection of tetrapeptide insertion variants by a recently established in vitro linker insertion mutagenesis technique. thirty-one distinct insertions were obtained, and their sigma fact ... | 2003 | 12700252 |
| involvement of the adc operon and manganese homeostasis in streptococcus gordonii biofilm formation. | pioneer oral bacteria, including streptococcus gordonii, initiate the formation of oral biofilms on tooth surfaces, which requires differential expression of genes that recognize unique environmental cues. an s. gordonii::tn917-lac biofilm-defective mutant was isolated by using an in vitro biofilm formation assay. subsequent inverse pcr and sequence analyses identified the transposon insertion to be near the 3' end of an open reading frame (orf) encoding a protein homologous to a streptococcus p ... | 2003 | 12700268 |
| role of 16s rrna helix 44 in ribosomal resistance to hygromycin b. | hygromycin b is an aminoglycoside antibiotic active against prokaryotic and eukaryotic ribosomes. ribosomal alterations in bacteria conferring resistance to hygromycin b have not been described, prompting us to use a single rrna allelic derivative of the gram-positive bacterium mycobacterium smegmatis for investigation of the molecular mechanisms involved in ribosomal resistance to hygromycin b in eubacteria. resistance mutations were found to localize exclusively in 16s rrna. the mutations obse ... | 2003 | 12709313 |
| the versatile thymine dna-glycosylase: a comparative characterization of the human, drosophila and fission yeast orthologs. | human thymine-dna glycosylase (tdg) is well known to excise thymine and uracil from g.t and g.u mismatches, respectively, and was therefore proposed to play a central role in the cellular defense against genetic mutation through spontaneous deamination of 5-methylcytosine and cytosine. in this study, we characterized two newly discovered orthologs of tdg, the drosophila melanogaster thd1p and the schizosaccharomyces pombe thp1p proteins, with an objective to address the function of this subfamil ... | 2003 | 12711670 |
| involvement of conserved asparagine and arginine residues from the n-terminal region in the catalytic mechanism of rat liver and trypanosoma cruzi tyrosine aminotransferases. | rat liver and trypanosoma cruzi tyrosine aminotransferases (tats) share over 40% sequence identity, but differ in their substrate specificities. to explore the molecular features related to these differences, comparative mutagenesis studies were conducted on full length t. cruzi tat and n-terminally truncated rat tat recombinant enzymes. the functionality of arg315 and arg417 in rat tat was investigated for comparison with the conserved arg292 and arg386 in aspartate and bacterial aromatic amino ... | 2003 | 12717026 |
| 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 ... | 2003 | 12719542 |
| 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 ... | 2003 | 12730374 |
| 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 ... | 2003 | 12732517 |
| 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 ... | 2003 | 12756229 |
| 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 ... | 2003 | 12756230 |
| 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 ... | 2003 | 12773376 |