| comparison of fungal 80 s ribosomes by cryo-em reveals diversity in structure and conformation of rrna expansion segments. | compared to the prokaryotic 70 s ribosome, the eukaryotic 80 s ribosome contains additional ribosomal proteins and extra segments of rrna, referred to as rrna expansion segments (es). these eukaryotic-specific rrna es are mainly on the periphery of the 80 s ribosome, as revealed by cryo-electron microscopy (cryo-em) studies, but their precise function is not known. to address the question of whether the rrna es are structurally conserved among 80 s ribosomes of different fungi we performed cryo- ... | 2007 | 17434183 |
| high-throughput identification of inhibitors of human mitochondrial peptide deformylase. | the human mitochondrial peptide deformylase (hspdf) provides a potential new target for broadly acting antiproliferative agents. to identify novel nonpeptidomimetic and nonhydroxamic acid-based inhibitors of hspdf, the authors have developed a high-throughput screening (hts) strategy using a fluorescence polarization (fp)-based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiprolifer ... | 2007 | 17435169 |
| analysis of the assembly profiles for mitochondrial- and nuclear-dna-encoded subunits into complex i. | complex i of the respiratory chain is composed of at least 45 subunits that assemble together at the mitochondrial inner membrane. defects in human complex i result in energy generation disorders and are also implicated in parkinson's disease and altered apoptotic signaling. the assembly of this complex is poorly understood and is complicated by its large size and its regulation by two genomes, with seven subunits encoded by mitochondrial dna (mtdna) and the remainder encoded by nuclear genes. h ... | 2007 | 17438127 |
| site-specific s-glutathiolation of mitochondrial nadh ubiquinone reductase. | the generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. overproduction of superoxide (o2*-) and o2*--derived oxidants changes the redox status of the mitochondrial gsh pool. an electron transport protein, mitochondrial complex i, is the major host of reactive/regulatory protein thiols. an important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfide vi ... | 2007 | 17444656 |
| structures of modified eef2 80s ribosome complexes reveal the role of gtp hydrolysis in translocation. | on the basis of kinetic data on ribosome protein synthesis, the mechanical energy for translocation of the mrna-trna complex is thought to be provided by gtp hydrolysis of an elongation factor (eef2 in eukaryotes, ef-g in bacteria). we have obtained cryo-em reconstructions of eukaryotic ribosomes complexed with adp-ribosylated eef2 (adpr-eef2), before and after gtp hydrolysis, providing a structural basis for analyzing the gtpase-coupled mechanism of translocation. using the adp-ribosyl group as ... | 2007 | 17446867 |
| enzymology and evolution of the pyruvate pathway to 2-oxobutyrate in methanocaldococcus jannaschii. | the archaeon methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme b biosyntheses. in each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. the genome sequence of m. jannaschii encodes two homologs of each large and small subunit that forms the hydrolyase, but the genes are not cotranscribed. the genes are more similar to each other than to p ... | 2007 | 17449626 |
| the 51-63 base pair of trna confers specificity for binding by ef-tu. | elongation factor tu (ef-tu) exhibits significant specificity for the different elongator trna bodies in order to offset its variable affinity to the esterified amino acid. three x-ray cocrystal structures reveal that while most of the contacts with the protein involve the phosphodiester backbone of trna, a single hydrogen bond is observed between the glu390 and the amino group of a guanine in the 51-63 base pair in the t-stem of trna. here we show that the glu390ala mutation of thermus thermoph ... | 2007 | 17449728 |
| mechanism for the ttdnaa-tt-oric cooperative interaction at high temperature and duplex opening at an unusual at-rich region in thermoanaerobacter tengcongensis. | thermoanaerobacter tengcongensis is an anaerobic low-gc thermophilic bacterium. to further elucidate the replication initiation of chromosomal dna at high temperature, the interaction between the replication initiator (ttdnaa) and the putative origin (tt-oric) in this thermophile was investigated. we found that efficient binding of ttdnaa to tt-oric at high temperature requires (i) at least two neighboring dnaa boxes, (ii) the specific feature of the ttdnaa domain iv and (iii) the self-oligomeri ... | 2007 | 17452366 |
| the outer membrane secretin pilq from neisseria meningitidis binds dna. | neisseria meningitidis is naturally competent for transformation throughout its growth cycle. transformation in neisserial species is coupled to the expression of type iv pili, which are present on the cell surface as bundled filamentous appendages, and are assembled, extruded and retracted by the pilus biogenesis components. during the initial phase of the transformation process, binding and uptake of dna takes place with entry through a presumed outer-membrane channel into the periplasm. this ... | 2007 | 17464074 |
| real-time footprinting of dna in the first kinetically significant intermediate in open complex formation by escherichia coli rna polymerase. | the architecture of cellular rna polymerases (rnaps) dictates that transcription can begin only after promoter dna bends into a deep channel and the start site nucleotide (+1) binds in the active site located on the channel floor. formation of this transcriptionally competent "open" complex (rp(o)) by escherichia coli rnap at the lambdap(r) promoter is greatly accelerated by dna upstream of base pair -47 (with respect to +1). here we report real-time hydroxyl radical (*oh) and potassium permanga ... | 2007 | 17470797 |
| post-transcriptional modification mapping in the clostridium acetobutylicum 16s rrna by mass spectrometry and reverse transcriptase assays. | post-transcriptional modifications in ribosomal rna are believed to fine-tune the rna functions. the present study describes the characterization of the post-transcriptional modifications in clostridium acetobutylicum 16s rrna, using high-pressure liquid chromatography (hplc) coupled to electrospray ionization mass spectrometry and reverse transcriptase assays. the combination of these techniques allowed the identification of eleven modified nucleosides, which were mapped onto the rrna sequence. ... | 2007 | 17478509 |
| structural elements defining elongation factor tu mediated suppression of codon ambiguity. | in most prokaryotes asn-trna(asn) and gln-trna(gln) are formed by amidation of aspartate and glutamate mischarged onto trna(asn) and trna(gln), respectively. coexistence in the organism of mischarged asp-trna(asn) and glu-trna(gln) and the homologous asn-trna(asn) and gln-trna(gln) does not, however, lead to erroneous incorporation of asp and glu into proteins, since ef-tu discriminates the misacylated trnas from the correctly charged ones. this property contrasts with the canonical function of ... | 2007 | 17478519 |
| fine-tuning of translation termination efficiency in saccharomyces cerevisiae involves two factors in close proximity to the exit tunnel of the ribosome. | in eukaryotes, release factors 1 and 3 (erf1 and erf3) are recruited to promote translation termination when a stop codon on the mrna enters at the ribosomal a-site. however, their overexpression increases termination efficiency only moderately, suggesting that other factors might be involved in the termination process. to determine such unknown components, we performed a genetic screen in saccharomyces cerevisiae that identified genes increasing termination efficiency when overexpressed. for th ... | 2007 | 17483428 |
| structural basis for functional mimicry of long-variable-arm trna by transfer-messenger rna. | tmrna and small protein b (smpb) are essential trans-translation system components. in the present study, we determined the crystal structure of smpb in complex with the entire trna domain of the tmrna from thermus thermophilus. overall, the ribonucleoprotein complex (trnp) mimics a long-variable-arm trna (class ii trna) in the canonical l-shaped tertiary structure. the tmrna terminus corresponds to the acceptor and t arms, or the upper part, of trna. on the other hand, the smpb protein simulate ... | 2007 | 17488812 |
| identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin. | capreomycin (cmn) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. resistance to these antibiotics in mycobacterium species arises due to mutations in the genes coding for the 16s or 23s rrna but can also arise due to mutations in a ... | 2007 | 17496129 |
| agrobacterium para/mind-like virc1 spatially coordinates early conjugative dna transfer reactions. | agrobacterium tumefaciens translocates t-dna through a polar virb/d4 type iv secretion (t4s) system. virc1, a factor required for efficient t-dna transfer, bears a deviant walker a and other sequence motifs characteristic of para and mind atpases. here, we show that virc1 promotes conjugative t-dna transfer by stimulating generation of multiple copies per cell of the t-dna substrate (t-complex) through pairwise interactions with the processing factors vird2 relaxase, virc2, and vird1. virc1 also ... | 2007 | 17505518 |
| cross-linked seca dimers are not functional in protein translocation. | the atpase seca is involved in post-translational protein translocation through the secy channel across the bacterial inner membrane. seca is a dimer that can dissociate into monomers with translocation activity. here, we have addressed whether dissociation of the seca dimer is required for translocation. we show that a dimer in which the two subunits are cross-linked by disulfide bridges is inactive in protein translocation, translocation atpase, and binding to a lipid bilayer. in contrast, upo ... | 2007 | 17511989 |
| rna chaperone activity of l1 ribosomal proteins: phylogenetic conservation and splicing inhibition. | rna chaperone activity is defined as the ability of proteins to either prevent rna from misfolding or to open up misfolded rna conformations. one-third of all large ribosomal subunit proteins from e. coli display this activity, with l1 exhibiting one of the highest activities. here, we demonstrate via the use of in vitro trans- and cis-splicing assays that the rna chaperone activity of l1 is conserved in all three domains of life. however, thermophilic archaeal l1 proteins do not display rna cha ... | 2007 | 17517772 |
| developing limited proteolysis and mass spectrometry for the characterization of ribosome topography. | an approach that combines limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (maldi-ms) has been developed to probe protease-accessible sites of ribosomal proteins from intact ribosomes. escherichia coli and thermus thermophilus 70s ribosomes were subjected to limited proteolysis using different proteases under strictly controlled conditions. intact ribosomal proteins and large proteolytic peptides were recovered and directly analyzed by maldi-ms, which allows ... | 2007 | 17521915 |
| interactions between the lipoprotein pilp and the secretin pilq in neisseria meningitidis. | neisseria meningitidis can be the causative agent of meningitis or septicemia. this bacterium expresses type iv pili, which mediate a variety of functions, including autoagglutination, twitching motility, biofilm formation, adherence, and dna uptake during transformation. the secretin pilq supports type iv pilus extrusion and retraction, but it also requires auxiliary proteins for its assembly and localization in the outer membrane. here we have studied the physical properties of the lipoprotein ... | 2007 | 17526700 |
| prediction of functional sites based on the fuzzy oil drop model. | a description of many biological processes requires knowledge of the 3-d structure of proteins and, in particular, the defined active site responsible for biological function. many proteins, the genes of which have been identified as the result of human genome sequencing, and which were synthesized experimentally, await identification of their biological activity. currently used methods do not always yield satisfactory results, and new algorithms need to be developed to recognize the localizatio ... | 2007 | 17530916 |
| the first agmatine/cadaverine aminopropyl transferase: biochemical and structural characterization of an enzyme involved in polyamine biosynthesis in the hyperthermophilic archaeon pyrococcus furiosus. | we report here the characterization of the first agmatine/cadaverine aminopropyl transferase (acapt), the enzyme responsible for polyamine biosynthesis from an archaeon. the gene pf0127 encoding acapt in the hyperthermophile pyrococcus furiosus was cloned and expressed in escherichia coli, and the recombinant protein was purified to homogeneity. p. furiosus acapt is a homodimer of 65 kda. the broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diamino ... | 2007 | 17545282 |
| collaboration between the clpb aaa+ remodeling protein and the dnak chaperone system. | clpb and hsp104, members of the aaa+ superfamily of proteins, protect cells from the devastating effects of protein inactivation and aggregation that arise after extreme heat stress. they exist as a hexameric ring and contain two nucleotide-binding sites per monomer. clpb and hsp104 are able to dissolve protein aggregates in conjunction with the dnak/hsp70 chaperone system, although the roles of the individual chaperones in disaggregation are not well understood. in the absence of the dnak/hsp70 ... | 2007 | 17545305 |
| long-range structural effects of a charcot-marie-tooth disease-causing mutation in human glycyl-trna synthetase. | functional expansion of specific trna synthetases in higher organisms is well documented. these additional functions may explain why dominant mutations in glycyl-trna synthetase (glyrs) and tyrosyl-trna synthetase cause charcot-marie-tooth (cmt) disease, the most common heritable disease of the peripheral nervous system. at least 10 disease-causing mutant alleles of glyrs have been annotated. these mutations scatter broadly across the primary sequence and have no apparent unifying connection. he ... | 2007 | 17545306 |
| new insights into the binding mode of coenzymes: structure of thermus thermophilus delta1-pyrroline-5-carboxylate dehydrogenase complexed with nadp+. | delta(1)-pyrroline-5-carboxylate dehydrogenase (p5cdh) is known to preferentially use nad(+) as a coenzyme. the k(cat) value of thermus thermophilus p5cdh (ttp5cdh) is four times lower for nadp(+) than for nad(+). the crystal structure of nadp(+)-bound ttp5cdh was solved in order to study the structure-activity relationships for the coenzymes. the binding mode of nadp(+) is essentially identical to that in the previously solved nad(+)-bound form, except for the regions around the additional 2'-p ... | 2007 | 17554163 |
| expression, purification, assay, and crystal structure of perdeuterated human arginase i. | arginase is a manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to yield l-ornithine and urea. in order to establish a foundation for future neutron diffraction studies that will provide conclusive structural information regarding proton/deuteron positions in enzyme-inhibitor complexes, we have expressed, purified, assayed, and determined the x-ray crystal structure of perdeuterated (i.e., fully deuterated) human arginase i complexed with 2(s)-amino-6-boronohexanoic acid (abh) ... | 2007 | 17562323 |
| structural basis for recruitment of tandem hotdog domains in acyl-coa thioesterase 7 and its role in inflammation. | acyl-coa thioesterases (acots) catalyze the hydrolysis of fatty acyl-coa to free fatty acid and coa and thereby regulate lipid metabolism and cellular signaling. we present a comprehensive structural and functional characterization of mouse acyl-coa thioesterase 7 (acot7). whereas prokaryotic homologues possess a single thioesterase domain, mammalian acot7 contains a pair of domains in tandem. we determined the crystal structures of both the n- and c-terminal domains of the mouse enzyme, and inf ... | 2007 | 17563367 |
| cloning, expression, purification, crystallization and preliminary x-ray analysis of thermus aquaticus succinyl-coa synthetase. | succinyl-coa synthetase (scs) is an enzyme of the citric acid cycle and is thus found in most species. to date, there are no structures available of scs from a thermophilic organism. to investigate how the enzyme adapts to higher temperatures, scs from thermus aquaticus was cloned, overexpressed, purified and crystallized. attempts to crystallize the enzyme were thwarted by proteolysis of the beta-subunit and preferential crystallization of the truncated form. crystals of full-length scs were gr ... | 2007 | 17565180 |
| preliminary x-ray crystallographic study of glucose dehydrogenase from thermus thermophilus hb8. | thermus thermophilus is an aerobic chemoorganotroph that has been found to grow anaerobically in the presence of nitrate. crystals of glucose dehydrogenase (gdh) from t. thermophilus hb8 belong to space group p2(1), with unit-cell parameters a = 36.90, b = 132.96, c = 60.78 a, beta = 97.2 degrees. preliminary studies and molecular-replacement calculations reveal that the asymmetric unit contains two monomers. | 2007 | 17565193 |
| crystal structure of saccharomyces cerevisiae 6-phosphogluconate dehydrogenase gnd1. | as the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6pgdh) is the main generator of cellular nadph. both thioredoxin reductase and glutathione reductase require nadph as the electron donor to reduce oxidized thioredoxin or glutathione (gssg). since thioredoxin and gsh are important antioxidants, it is not surprising that 6pgdh plays a critical role in protecting cells from oxidative stress. furthermore the activity of 6pgdh is associated with several human dis ... | 2007 | 17570834 |
| structural basis for recognition of cognate trna by tyrosyl-trna synthetase from three kingdoms. | the specific aminoacylation of trna by tyrosyl-trna synthetases (tyrrss) relies on the identity determinants in the cognate trna(tyr)s. we have determined the crystal structure of saccharomyces cerevisiae tyrrs (scetyrrs) complexed with a tyr-amp analog and the native trna(tyr)(gpsia). structural information for tyrrs-trna(tyr) complexes is now full-line for three kingdoms. because the archaeal/eukaryotic tyrrss-trna(tyr)s pairs do not cross-react with their bacterial counterparts, the recogniti ... | 2007 | 17576676 |
| a loop loop interaction and a k-turn motif located in the lysine aptamer domain are important for the riboswitch gene regulation control. | the lysine riboswitch is associated to the lysc gene in bacillus subtilis, and the binding of lysine modulates the rna structure to allow the formation of an intrinsic terminator presumably involved in transcription attenuation. the complex secondary structure of the lysine riboswitch aptamer is organized around a five-way junction that undergoes structural changes upon ligand binding. using single-round transcription assays, we show that a loop-loop interaction is important for lysine-induced t ... | 2007 | 17585050 |
| characterization and mechanistic studies of type ii isopentenyl diphosphate:dimethylallyl diphosphate isomerase from staphylococcus aureus. | the recently identified type ii isopentenyl diphosphate (ipp):dimethylallyl diphosphate (dmapp) isomerase (idi-2) is a flavoenzyme that requires fmn and nad(p)h for activity. idi-2 is an essential enzyme for the biosynthesis of isoprenoids in several pathogenic bacteria including staphylococcus aureus, streptococcus pneumoniae, and enterococcus faecalis, and thus is considered as a potential new drug target to battle bacterial infections. one notable feature of the idi-2 reaction is that there i ... | 2007 | 17585782 |
| protein aq_1862 from the hyperthermophilic bacterium aquifex aeolicus is a porin and contains two conductance pathways of different selectivity. | the "hypothetical protein" aq_1862 was isolated from the membrane fraction of aquifex aeolicus and identified as the major porin. in experiments with one conducting unit (molecule) a conductance of 1.4 ns was observed in 0.1 m kcl at ph 7.5. this stable (basic) conductance was superimposed by conductance fluctuations of approximately 0.25 ns. because both events were always observed simultaneously, it is suggested that they are caused by the same molecular entity. nonetheless they show very diff ... | 2007 | 17586565 |
| the 2.2 a resolution crystal structure of bacillus cereus nif3-family protein yqfo reveals a conserved dimetal-binding motif and a regulatory domain. | yqfo of bacillus cereus is a member of the widespread nif3 family of proteins, which has been highlighted as an important target for structural genomics. the n- and c-terminal domains are conserved across the family and contain a dimetal-binding motif in a putative active site. yqfo contains an insert in the middle of the protein, present in a minority of bacterial family members. the structure of yqfo was determined at a resolution of 2.2 a and reveals conservation of the putative active site. ... | 2007 | 17586767 |
| crystal structures of tm0549 and ne1324--two orthologs of e. coli ahas isozyme iii small regulatory subunit. | crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase iii (ahas, ec 2.2.1.6) from thermotoga maritima (tm0549) and nitrosomonas europea (ne1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 a and 2.5 a, respectively. tm0549 and ne1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. each protein contains a c-terminal domain ... | 2007 | 17586771 |
| direct localization of the 51 and 24 kda subunits of mitochondrial complex i by three-dimensional difference imaging. | complex i is the largest complex in the respiratory chain, and the least understood. we have determined the 3d structure of complex i from yarrowia lipolytica lacking the flavoprotein part of the n-module, which consists of the 51 kda (nubm) and the 24 kda (nuhm) subunits. the reconstruction was determined by 3d electron microscopy of single particles. a comparison to our earlier reconstruction of the complete y. lipolytica complex i clearly assigns the two flavoprotein subunits to an outer lobe ... | 2007 | 17591445 |
| testing constraints on rrna bases that make nonsequence-specific contacts with the codon-anticodon complex in the ribosomal a site. | during protein synthesis, interactions between the decoding center of the ribosome and the codon-anticodon complexes maintain translation accuracy. correct aminoacyl-trnas induce the ribosome to shift into a "closed" conformation that both blocks trna dissociation and accelerates the process of trna acceptance. as part of the ribosomal recognition of cognate trnas, the rrna nucleotides g530 and a1492 form a hydrogen-bonded pair that interacts with the middle position of the codon.anticodon compl ... | 2007 | 17592040 |
| geranylgeranyl diphosphate synthase in fission yeast is a heteromer of farnesyl diphosphate synthase (fps), fps1, and an fps-like protein, spo9, essential for sporulation. | both farnesyl diphosphate synthase (fps) and geranylgeranyl diphosphate synthase (ggps) are key enzymes in the synthesis of various isoprenoid-containing compounds and proteins. here, we describe two novel schizosaccharomyces pombe genes, fps1(+) and spo9(+), whose products are similar to fps in primary structure, but whose functions differ from one another. fps1 is essential for vegetative growth, whereas, a spo9 null mutant exhibits temperature-sensitive growth. expression of fps1(+), but not ... | 2007 | 17596513 |
| modeling of phosphomethyl pyrimidine kinase from leptospira interrogans serovar lai strain 56601. | many microorganisms, as well as plants and fungi, synthesize thiamin, but vertebrates do not produce it. phosphomethyl pyrimidine kinase is an enzyme involved in an intermediary step of thiamin biosynthesis from purine molecules. this enzyme is absent in humans. thus, it is a potential chemotherapeutic target for antileptospiral treatment. structure of this enzyme from leptospira interrogans serovar lai strain 56601 has not yet been elucidated. we used the structural template of phosphomethyl py ... | 2006 | 17597880 |
| paradigm development: comparative and predictive 3d modeling of hiv-1 virion infectivity factor (vif). | obtaining structural information about vif is of interest for several reasons that include the study of the interaction of vif with apobec3g, a resistance factor. vif is a potential drug target and its function is essential for the hiv-1 infectivity process. to study vif mechanism of action, we need to decipher its structure. pivotal in this approach is the painstaking prediction of its protein structure. the three-dimensional (3d) crystal structure for vif has not been established. in order to ... | 2006 | 17597910 |
| use of a dominant rpsl allele conferring streptomycin dependence for positive and negative selection in thermus thermophilus. | a spontaneous rpsl mutant of thermus thermophilus was isolated in a search for new selection markers for this organism. this new allele, named rpsl1, encodes a k47r/k57e double mutant s12 ribosomal protein that confers a streptomycin-dependent (sd) phenotype to t. thermophilus. models built on the available three-dimensional structures of the 30s ribosomal subunit revealed that the k47r mutation directly affects the streptomycin binding site on s12, whereas the k57e does not apparently affect th ... | 2007 | 17601820 |
| mechanistic studies of the long chain acyl-coa synthetase faa1p from saccharomyces cerevisiae. | long chain acyl-coa synthetase (acsl; fatty acid coa ligase: amp forming; ec 6.2.1.3) catalyzes the formation of acyl-coa through a process, which requires fatty acid, atp and coenzymea as substrates. in the yeast saccharomyces cerevisiae the principal acsl is faa1p (encoded by the faa1 gene). the preferred substrates for this enzyme are cis-monounsaturated long chain fatty acids. our previous work has shown faa1p is a principal component of a fatty acid transport/activation complex that also in ... | 2007 | 17604220 |
| thermostability of proteins: role of metal binding and ph on the stability of the dinuclear cua site of thermus thermophilus. | the dinuclear copper center (ttcua) forming the electron entry site in the subunit ii of the cytochrome c oxidase in thermus thermophilus shows high stability toward thermal as well as denaturant-induced unfolding of the protein at ambient ph. we have studied the effect of ph on the stability of the holo-protein as well as of the apo-protein by uv-visible absorption, far-uv, and visible circular dichroism spectroscopy. the results show that the holo-protein both in the native mixed-valence state ... | 2007 | 17604317 |
| structural characterization of the ribosome maturation protein, rimm. | the rimm protein has been implicated in the maturation of the 30s ribosomal subunit. it binds to ribosomal protein s19, located in the head domain of the 30s subunit. multiple sequence alignments predicted that rimm possesses two domains in its n- and c-terminal regions. in the present study, we have produced thermus thermophilus rimm in both the full-length form (162 residues) and its n-terminal fragment, spanning residues 1 to 85, as soluble proteins in escherichia coli and have performed stru ... | 2007 | 17616598 |
| use of an escherichia coli recombinant producing thermostable polyphosphate kinase as an atp regenerator to produce fructose 1,6-diphosphate. | heat-treated escherichia coli producing thermus polyphosphate kinase regenerated atp by using exogenous polyphosphate. this recombinant could be used as a platform to produce valuable compounds in combination with thermostable phosphorylating or energy-requiring enzymes. in this work, we demonstrated the production of fructose 1,6-diphosphate from fructose and polyphosphate. | 2007 | 17616610 |
| crystallization and preliminary x-ray analysis of the oxygenase component (hpab) of 4-hydroxyphenylacetate 3-monooxygenase from thermus thermophilus hb8. | the 4-hydroxyphenylacetate (4hpa) 3-monooxygenase enzyme catalyzes the hydroxylation of 4hpa to 3,4-dihydroxyphenylacetate in the initial step of the degradation pathway of 4hpa. this enzyme consists of two components: an oxygenase (hpab) and a reductase (hpac). hpab hydroxylates 4hpa using an oxygen molecule and a reduced flavin, which is supplied by hpac. hpab from thermus thermophilus hb8 was overexpressed in escherichia coli and crystallized. crystals of hpab were grown in 0.4 m 1,6-hexanedi ... | 2007 | 17620709 |
| crystallization and preliminary x-ray crystallographic study of a putative aspartyl-trna synthetase from the crenarchaeon sulfolobus tokodaii strain 7. | genome analysis suggests that the aspartyl-trna synthetase of the crenarchaeon sulfolobus tokodaii strain 7 belongs to the nondiscriminating type that is believed to catalyze aspartylation of trna(asp) and trna(asn). this protein has been overexpressed in escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method from 100 mm sodium hepes buffer ph 7.5 containing 100 mm nacl and 1.6 m (nh4)2so4 as the crystallizing reagent. diffraction data were collected to 2.3 a ... | 2007 | 17620724 |
| human endonuclease viii-like (neil) proteins in the giant dna mimivirus. | endonuclease viii (nei), which recognizes and repairs oxidized pyrimidines in the base excision repair (ber) pathway, is sparsely distributed among both the prokaryotes and eukaryotes. recently, we and others identified three homologs of escherichia coli endonuclease viii-like (neil) proteins in humans. here, we report identification of human neil homologs in mimivirus, a giant dna virus that infects acanthamoeba. characterization of the two mimiviral homologs, mvnei1 and mvnei2, showed that the ... | 2007 | 17627905 |
| molecular breeding of polymerases for amplification of ancient dna. | in the absence of repair, lesions accumulate in dna. thus, dna persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged. we describe a strategy for the recovery of genetic information from damaged dna. by molecular breeding of polymerase genes from the genus thermus (taq (thermus aquaticus), tth (thermus thermophilus) and tfl (thermus flavus)) and compartmentalized self-replication selection, we have evolved polymerases that can extend single, double ... | 2007 | 17632524 |
| effects of the deletion of the escherichia coli frataxin homologue cyay on the respiratory nadh:ubiquinone oxidoreductase. | frataxin is discussed as involved in the biogenesis of iron-sulfur clusters. recently it was discovered that a frataxin homologue is a structural component of the respiratory nadh:ubiquinone oxidoreductase (complex i) in thermus thermophilus. it was not clear whether frataxin is in general a component of complex i from bacteria. the escherichia coli homologue of frataxin is coined cyay. | 2007 | 17650323 |
| changes in the conformation of 5s rrna cause alterations in principal functions of the ribosomal nanomachine. | 5s rrna is an integral component of the large ribosomal subunit in virtually all living organisms. polyamine binding to 5s rrna was investigated by cross-linking of n1-azidobenzamidino (aba)-spermine to naked 5s rrna or 50s ribosomal subunits and whole ribosomes from escherichia coli cells. aba-spermine cross-linking sites were kinetically measured and their positions in 5s rrna were localized by primer extension analysis. helices iii and v, and loops a, c, d and e in naked 5s rrna were found to ... | 2007 | 17652323 |
| a comparative analysis of the triloops in all high-resolution rna structures reveals sequence structure relationships. | despite an increasing number of experimentally determined rna structures, the gap between the number of structures and that of rna families is still growing. to overcome this limitation, efficient and reliable rna modeling methodologies must be developed. in order to reach this goal, here, we show how triloop sequence-structure relationships have been inferred through a systematic analysis of all triloops found in available high-resolution structures. the structural annotation of all triloops al ... | 2007 | 17652406 |
| biosynthesis of isoprenoids in plants: structure of the 2c-methyl-d-erithrytol 2,4-cyclodiphosphate synthase from arabidopsis thaliana. comparison with the bacterial enzymes. | the x-ray crystal structure of the 2c-methyl-d-erythritol 2,4-cyclodiphosphate synthase (mcs) from arabidopsis thaliana has been solved at 2.3 a resolution in complex with a cytidine-5-monophosphate (cmp) molecule. this is the first structure determined of an mcs enzyme from a plant. major differences between the a. thaliana and bacterial mcs structures are found in the large molecular cavity that forms between subunits and involve residues that are highly conserved among plants. in some bacteri ... | 2007 | 17660251 |
| tertiary structure and function of an rna motif required for plant vascular entry to initiate systemic trafficking. | vascular entry is a decisive step for the initiation of long-distance movement of infectious and endogenous rnas, silencing signals and developmental/defense signals in plants. however, the mechanisms remain poorly understood. we used potato spindle tuber viroid (pstvd) as a model to investigate the direct role of the rna itself in vascular entry. we report here the identification of an rna motif that is required for pstvd to traffic from nonvascular into the vascular tissue phloem to initiate s ... | 2007 | 17660743 |
| negamycin binds to the wall of the nascent chain exit tunnel of the 50s ribosomal subunit. | negamycin, a small-molecule inhibitor of protein synthesis, binds the haloarcula marismortui 50s ribosomal subunit at a single site formed by highly conserved rna nucleotides near the cytosolic end of the nascent chain exit tunnel. the mechanism of antibiotic action and the function of this unexplored tunnel region remain intriguingly elusive. | 2007 | 17664317 |
| crystallization and preliminary x-ray diffraction analysis of a soluble domain of the putative zinc transporter czrb from thermus thermophilus. | czrb is a putative zinc transporter from thermus thermophilus. the protein is proposed to consist of a hexahelical transmembrane domain with a cytosolic extramembranal c-terminus. the latter 92-residue fragment may be expressed free and may function independently of the full-length integral membrane protein. a 6xhis-tagged form of the water-soluble fragment has been overexpressed in escherichia coli and diffraction-quality crystals of the tagged and tag-free variants have been grown. preliminary ... | 2007 | 17671365 |
| crystallization and preliminary x-ray diffraction analysis of the cytosolic domain of the mg2+ transporter mgte. | the mgte family of mg(2+) transporters is ubiquitously conserved in all domains of life. the cytosolic domains of the mgte mg(2+) transporters include a cystathionine-beta-synthase (cbs) domain which is known to play a regulatory function in transporter proteins. the cytosolic domain of mgte from thermus thermophilus was overexpressed, purified and crystallized in the presence and absence of mg(2+). the crystals formed in the presence of mg(2+) diffracted x-rays to 2.3 a resolution using synchro ... | 2007 | 17671366 |
| crystallization and preliminary x-ray diffraction analysis of the full-length mg2+ transporter mgte. | the mgte family of mg(2+) transporters are ubiquitously conserved in all three domains. the genes encoding full-length mgte from seven different species were cloned. three of the seven mgte transporters were overexpressed and purified for use in crystallization trials. only thermus thermophilus mgte was successfully crystallized using the sitting-drop vapour-diffusion method. selenomethionine-substituted (semet) crystals were obtained by cross-microseeding using the native microcrystals. the sem ... | 2007 | 17671367 |
| crystallization and preliminary crystallographic analysis of hygromycin b phosphotransferase from escherichia coli. | aminoglycoside antibiotics, such as hygromycin, kanamycin, neomycin, spectinomycin and streptomycin, inhibit protein synthesis by acting on bacterial and eukaryotic ribosomes. hygromycin b phosphotransferase (hph; ec 2.7.1.119) converts hygromycin b to 7''-o-phosphohygromycin using a phosphate moiety from atp, resulting in the loss of its cell-killing activity. the hph protein has been crystallized for the first time using a thermostable mutant and the hanging-drop vapour-diffusion method. the c ... | 2007 | 17671368 |
| complete genomic sequence and mass spectrometric analysis of highly diverse, atypical bacillus thuringiensis phage 0305phi8-36. | to investigate the apparent genomic complexity of long-genome bacteriophages, we have sequenced the 218,948-bp genome (6479-bp terminal repeat), and identified the virion proteins (55), of bacillus thuringiensis bacteriophage 0305phi8-36. phage 0305phi8-36 is an atypical myovirus with three large curly tail fibers. an accurate mode of dna pyrosequencing was used to sequence the genome and mass spectrometry was used to accomplish the comprehensive virion protein survey. advanced informatic techni ... | 2007 | 17673272 |
| the archaeon methanosarcina acetivorans contains a protein disulfide reductase with an iron-sulfur cluster. | methanosarcina acetivorans, a strictly anaerobic methane-producing species belonging to the domain archaea, contains a gene cluster annotated with homologs encoding oxidative stress proteins. one of the genes (ma3736) is annotated as a gene encoding an uncharacterized carboxymuconolactone decarboxylase, an enzyme required for aerobic growth with aromatic compounds by species in the domain bacteria. methane-producing species are not known to utilize aromatic compounds, suggesting that ma3736 is i ... | 2007 | 17675382 |
| crystal structure of the escherichia coli regulator of sigma70, rsd, in complex with sigma70 domain 4. | the escherichia coli rsd protein binds tightly and specifically to the rna polymerase (rnap) sigma(70) factor. rsd plays a role in alternative sigma factor-dependent transcription by biasing the competition between sigma(70) and alternative sigma factors for the available core rnap. here, we determined the 2.6 a-resolution x-ray crystal structure of rsd bound to sigma(70) domain 4 (sigma(70)(4)), the primary determinant for rsd binding within sigma(70). the structure reveals that rsd binding int ... | 2007 | 17681541 |
| discovery of novel dna gyrase inhibitors by high-throughput virtual screening. | the bacterial type ii topoisomerases dna gyrase and topoisomerase iv are validated targets for clinically useful quinolone antimicrobial drugs. a significant limitation to widely utilized quinolone inhibitors is the emergence of drug-resistant bacteria due to an altered dna gyrase. to address this problem, we have used structure-based molecular docking to identify novel drug-like small molecules that target sites distinct from those targeted by quinolone inhibitors. a chemical ligand database co ... | 2007 | 17682095 |
| analysis of a nuclease activity of catalytic domain of thermus thermophilus muts2 by high-accuracy mass spectrometry. | electrospray ionization with fourier-transform ion cyclotron resonance mass spectrometry (esi-ft icr ms) is a powerful tool for analyzing the precise structural features of biopolymers, including oligonucleotides. here, we described the detailed characterization of a newly discovered nuclease activity of the c-terminal domain of thermus thermophilus muts2 (ttmuts2). using this method, the length, nucleotide content and nature of the 5'- and 3'-termini of the product oligonucleotides were accurat ... | 2007 | 17686785 |
| hitting bacteria at the heart of the central dogma: sequence-specific inhibition. | abstract: an important objective in developing new drugs is the achievement of high specificity to maximize curing effect and minimize side-effects, and high specificity is an integral part of the antisense approach. the antisense techniques have been extensively developed from the application of simple long, regular antisense rna (asrna) molecules to highly modified versions conferring resistance to nucleases, stability of hybrid formation and other beneficial characteristics, though still pres ... | 2007 | 17692125 |
| identification of indole derivatives as self-growth inhibitors of symbiobacterium thermophilum, a unique bacterium whose growth depends on coculture with a bacillus sp. | symbiobacterium thermophilum is a syntrophic bacterium whose growth depends on coculture with a bacillus sp. recently, we discovered that co(2) generated by bacillus is the major inducer for the growth of s. thermophilum; however, the evidence suggested that an additional element is required for its full growth. here, we studied the self-growth-inhibitory substances produced by s. thermophilum. we succeeded in purifying two substances from an ether extract of the culture supernatant of s. thermo ... | 2007 | 17693561 |
| type i and type ii fatty acid biosynthesis in eimeria tenella: enoyl reductase activity and structure. | apicomplexan parasites of the genus eimeria are the major causative agent of avian coccidiosis, leading to high economic losses in the poultry industry. recent results show that eimeria tenella harbours an apicoplast organelle, and that a key biosynthetic enzyme, enoyl reductase, is located in this organelle. in related parasites, enoyl reductase is one component of a type ii fatty acid synthase (fas) and has proven to be an attractive target for antimicrobial compounds. we cloned and expressed ... | 2007 | 17697396 |
| a functional interaction of smpb with tmrna for determination of the resuming point of trans-translation. | in trans-translation, transfer-messenger rna (tmrna), possessing a dual function as a trna and an mrna, relieves a stalled translation on the ribosome with the help of smpb. here, we established an in vitro system using escherichia coli translation and trans-translation factors to evaluate two steps of trans-translation, peptidyl transfer from peptidyl-trna to alanyl-tmrna and translation of the resume codon on tmrna. using this system, the effects of several mutations upstream of the tag-encodi ... | 2007 | 17698641 |
| conformations of flanking bases in hiv-1 rna dis kissing complexes studied by molecular dynamics. | explicit solvent molecular dynamics simulations (in total almost 800 ns including locally enhanced sampling runs) were applied with different ion conditions and with two force fields (amber and charmm) to characterize typical geometries adopted by the flanking bases in the rna kissing-loop complexes. we focus on flanking base positions in multiple x-ray and nmr structures of hiv-1 dis kissing complexes and kissing complex from the large ribosomal subunit of haloarcula marismortui. an initial x-r ... | 2007 | 17704156 |
| enzymes involved in dna ligation and end-healing in the radioresistant bacterium deinococcus radiodurans. | enzymes involved in dna metabolic events of the highly radioresistant bacterium deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the deinococcus radiodurans genome after extremely high doses of gamma-irradiation. although several deinococcus radiodurans dna repair enzymes have been characterised, no biochemical data is available for dna ligation and dna endhealing enzymes of deinococcus radiodurans so far. dna ligases are necessary to seal broke ... | 2007 | 17705817 |
| allosteric control of the rna polymerase by the elongation factor rfah. | efficient transcription of long polycistronic operons in bacteria frequently relies on accessory proteins but their molecular mechanisms remain obscure. rfah is a cellular elongation factor that acts as a polarity suppressor by increasing rna polymerase (rnap) processivity. in this work, we provide evidence that rfah acts by reducing transcriptional pausing at certain positions rather than by accelerating rnap at all sites. we show that 'fast' rnap variants are characterized by pause-free rna ch ... | 2007 | 17711918 |
| messenger rna conformations in the ribosomal e site revealed by x-ray crystallography. | a comparison of messenger rna in x-ray crystal structures of 70s ribosomal complexes in the initiation, post-initiation and elongation states of translation shows distinct conformational differences in the exit (e) codon. here, we present structural evidence indicating that, after the initiation event, the e codon nucleotides relax and form a classical a-helical conformation. this conformation is similar to that of the p and a codons, and is favourable for establishing watson-crick interactions ... | 2007 | 17721443 |
| human tryptophanyl-trna synthetase is switched to a trna-dependent mode for tryptophan activation by mutations at v85 and i311. | for most aminoacyl-trna synthetases (aars), their cognate trna is not obligatory to catalyze amino acid activation, with the exception of four class i (aars): arginyl-trna synthetase, glutamyl-trna synthetase, glutaminyl-trna synthetase and class i lysyl-trna synthetase. furthermore, for arginyl-, glutamyl- and glutaminyl-trna synthetase, the integrated 3' end of the trna is necessary to activate the atp-ppi exchange reaction. tryptophanyl-trna synthetase is a class i aars that catalyzes tryptop ... | 2007 | 17726052 |
| anticodon recognition and discrimination by the alpha-helix cage domain of class i lysyl-trna synthetase. | aminoacyl-trna synthetases are normally found in one of two mutually exclusive structural classes, the only known exception being lysyl-trna synthetase which exists in both classes i (lysrs1) and ii (lysrs2). differences in trna acceptor stem recognition between lysrs1 and lysrs2 do not drastically impact cellular aminoacylation levels, focusing attention on the mechanism of trna anticodon recognition by lysrs1. on the basis of structure-based sequence alignments, seven trnalys anticodon variant ... | 2007 | 17760422 |
| redox-dependent change of nucleotide affinity to the active site of the mammalian complex i. | a very potent and specific inhibitor of mitochondrial nadh:ubiquinone oxidoreductase (complex i), a derivative of nadh (nadh-oh) has recently been discovered (kotlyar, a. b., karliner, j. s., and cecchini, g. (2005) febs lett. 579, 4861-4866). here we present a quantitative analysis of the interaction of nadh-oh and other nucleotides with oxidized and reduced complex i in tightly coupled submitochondrial particles. both the rate of the nadh-oh binding and its affinity to complex i are strongly d ... | 2007 | 17760425 |
| structure-based design of robust glucose biosensors using a thermotoga maritima periplasmic glucose-binding protein. | we report the design and engineering of a robust, reagentless fluorescent glucose biosensor based on the periplasmic glucose-binding protein obtained from thermotoga maritima (tmgbp). the gene for this protein was cloned from genomic dna and overexpressed in escherichia coli, the identity of its cognate sugar was confirmed, ligand binding was studied, and the structure of its glucose complex was solved to 1.7 angstrom resolution by x-ray crystallography. tmgbp is specific for glucose and exhibit ... | 2007 | 17766373 |
| analysis of promoter targets for escherichia coli transcription elongation factor grea in vivo and in vitro. | transcription elongation factor grea induces nucleolytic activity of bacterial rna polymerase (rnap). in vitro, transcript cleavage by grea contributes to transcription efficiency by (i) suppressing pauses and arrests, (ii) stimulating rnap promoter escape, and (iii) enhancing transcription fidelity. however, it is unclear which of these functions is (are) most relevant in vivo. by comparing global gene expression profiles of escherichia coli strains lacking gre factors and strains expressing ei ... | 2007 | 17766423 |
| an aminoacyl-trna synthetase:elongation factor complex for substrate channeling in archaeal translation. | translation requires the specific attachment of amino acids to trnas by aminoacyl-trna synthetases (aarss) and the subsequent delivery of aminoacyl-trnas to the ribosome by elongation factor 1 alpha (ef-1alpha). interactions between ef-1alpha and various aarss have been described in eukaryotes, but the role of these complexes remains unclear. to investigate possible interactions between ef-1alpha and other cellular components, a yeast two-hybrid screen was performed for the archaeon methanotherm ... | 2007 | 17766929 |
| purification, crystallization and preliminary x-ray characterization of a human mitochondrial phenylalanyl-trna synthetase. | human monomeric mitochondrial phenylalanyl-trna synthetase (mitphers) is an enzyme that catalyzes the charging of trna with the cognate amino acid phenylalanine. human mitphers is a chimera of the bacterial alpha-subunit of phers and the b8 domain of its beta-subunit. together, the alpha-subunit and the 'rnp-domain' (b8 domain) at the c-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. the recombinant human mitphers was purified to homogeneity and c ... | 2007 | 17768348 |
| crystallization and preliminary x-ray crystallographic analysis of a 40 kda n-terminal fragment of the yeast prion-remodeling factor hsp104. | a 40 kda n-terminal fragment of saccharomyces cerevisiae hsp104 was crystallized in two different crystal forms. native 1 diffracted to 2.6 a resolution and belonged to space group p2(1)2(1)2(1), with unit-cell parameters a = 66.6, b = 75.8, c = 235.7 a. native 2 diffracted to 2.9 a resolution and belonged to space group p6(1)22 or p6(5)22, with unit-cell parameters a = 179.1, b = 179.1, c = 69.7 a. this is the first report of the crystallization of a eukaryotic member of the hsp100 family of mo ... | 2007 | 17768355 |
| purification, crystallization and preliminary x-ray analysis of the fumarylacetoacetase family member ttha0809 from thermus thermophilus hb8. | fumarylacetoacetase catalyzes the final step of tyrosine and phenylalanine catabolism. a recombinant form of the fumarylacetoacetase family member ttha0809 from thermus thermophilus hb8 has been crystallized by the oil-microbatch method using sodium chloride as a precipitating agent. the crystals belong to the monoclinic space group p2(1), with unit-cell parameters a = 93.3, b = 73.4, c = 122.6 a, beta = 111.8 degrees. the crystals are most likely to contain two dimers in the asymmetric unit, wi ... | 2007 | 17768357 |
| ribosome biogenesis and the translation process in escherichia coli. | translation, the decoding of mrna into protein, is the third and final element of the central dogma. the ribosome, a nucleoprotein particle, is responsible and essential for this process. the bacterial ribosome consists of three rrna molecules and approximately 55 proteins, components that are put together in an intricate and tightly regulated way. when finally matured, the quality of the particle, as well as the amount of active ribosomes, must be checked. the focus of this review is ribosome b ... | 2007 | 17804668 |
| nmr analysis of [methyl-13c]methionine uvrb from bacillus caldotenax reveals uvrb-domain 4 heterodimer formation in solution. | uvrb is a central dna damage recognition protein involved in bacterial nucleotide excision repair. structural information has been limited by the apparent disorder of the c-terminal domain 4 in crystal structures of intact uvrb; in solution, the isolated domain 4 is found to form a helix-loop-helix dimer. in order to gain insight into the behavior of uvrb in solution, we have performed nmr studies on [methyl-13c]methionine-labeled uvrb from bacillus caldotenax (molecular mass=75 kda). the 13 met ... | 2007 | 17822711 |
| elongation factor 1a mediates the specificity of mitochondrial trna import in t. brucei. | mitochondrial trna import is widespread in eukaryotes. yet, the mechanism that determines its specificity is unknown. previous in vivo experiments using the trnas(met), trna(ile) and trna(lys) have suggested that the t-stem nucleotide pair 51:63 is the main localization determinant of trnas in trypanosoma brucei. in the cytosol-specific initiator trna(met), this nucleotide pair is identical to the main antideterminant that prevents interaction with cytosolic elongation factor (eef1a). here we sh ... | 2007 | 17853889 |
| structural and kinetic characterization of quinolinate phosphoribosyltransferase (hqprtase) from homo sapiens. | human quinolinate phosphoribosyltransferase (ec 2.4.2.19) (hqprtase) is a member of the type ii phosphoribosyltransferase family involved in the catabolism of quinolinic acid (qa). it catalyses the formation of nicotinic acid mononucleotide from quinolinic acid, which involves a phosphoribosyl transfer reaction followed by decarboxylation. hqprtase has been implicated in a number of neurological conditions and in order to study it further, we have carried out structural and kinetic studies on re ... | 2007 | 17868694 |
| substrate specificity and properties of the escherichia coli 16s rrna methyltransferase, rsme. | the small ribosome subunit of escherichia coli contains 10 base-methylated sites distributed in important functional regions. at present, seven enzymes responsible for methylation of eight bases are known, but most of them have not been well characterized. one of these enzymes, rsme, was recently identified and shown to specifically methylate u1498. here we describe the enzymatic properties and substrate specificity of rsme. the enzyme forms dimers in solution and is most active in the presence ... | 2007 | 17872509 |
| conformational energy and structure in canonical and noncanonical forms of trna determined by temperature analysis of the rate of s(4)u8-c13 photocrosslinking. | bacterial trnas frequently have 4-thiouridine (s(4)u) modification at position 8, which is adjacent to the c13-g22-m(7)g46 base triple in the elbow region of the trna tertiary structure. irradiation with light in the uva range induces an efficient photocrosslink between s(4)u8 and c13. the temperature dependence of the rate constants for photocrosslinking between the s(4)u8 and c13 has been used to investigate the trna conformational energy and structure in escherichia coli trna(val), trna(phe), ... | 2007 | 17872510 |
| phosphopantetheine adenylyltransferase from escherichia coli: investigation of the kinetic mechanism and role in regulation of coenzyme a biosynthesis. | phosphopantetheine adenylyltransferase (ppat) from escherichia coli is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme a (coa) biosynthesis and is a target for antibacterial drug discovery. the enzyme utilizes mg-atp and phosphopantetheine (php) to generate dephospho-coa (dpcoa) and pyrophosphate. when overexpressed in e. coli, ppat copurifies with tightly bound coa, suggesting a feedback inhibitory role for this cofactor. using an enzyme-coupled assay for the forwa ... | 2007 | 17873050 |
| pathogenic mechanism of a human mitochondrial trnaphe mutation associated with myoclonic epilepsy with ragged red fibers syndrome. | human mitochondrial trna (hmt-trna) mutations are associated with a variety of diseases including mitochondrial myopathies, diabetes, encephalopathies, and deafness. because the current understanding of the precise molecular mechanisms of these mutations is limited, there is no efficient method to treat their associated mitochondrial diseases. here, we use a variety of known mutations in hmt-trna(phe) to investigate the mechanisms that lead to malfunctions. we tested the impact of hmt-trna(phe) ... | 2007 | 17878308 |
| human ribosomal protein s13 regulates expression of its own gene at the splicing step by a feedback mechanism. | the expression of ribosomal protein (rp) genes is regulated at multiple levels. in yeast, two genes are autoregulated by feedback effects of the protein on pre-mrna splicing. here, we have investigated whether similar mechanisms occur in eukaryotes with more complicated and highly regulated splicing patterns. comparisons of the sequences of ribosomal protein s13 gene (rps13) among mammals and birds revealed that intron 1 is more conserved than the other introns. transfection of hek 293 cells wit ... | 2007 | 17881366 |
| role of hsp104 in the propagation and inheritance of the [het-s] prion. | the chaperones of the clpb/hsp100 family play a central role in thermotolerance in bacteria, plants, and fungi by ensuring solubilization of heat-induced protein aggregates. in addition in yeast, hsp104 was found to be required for prion propagation. herein, we analyze the role of podospora anserina hsp104 (pahsp104) in the formation and propagation of the [het-s] prion. we show that deltapahsp104 strains propagate [het-s], making [het-s] the first native fungal prion to be propagated in the abs ... | 2007 | 17881723 |
| saturation mutagenesis of a +1 programmed frameshift-inducing mrna sequence derived from a yeast retrotransposon. | errors during the process of translating mrna information into protein products occur infrequently. frameshift errors occur less frequently than other types of errors, suggesting that the translational machinery has more robust mechanisms for precluding that kind of error. despite these mechanisms, mrna sequences have evolved that increase the frequency up to 10,000-fold. these sequences, termed programmed frameshift sites, usually consist of a heptameric nucleotide sequence, at which the change ... | 2007 | 17881742 |
| dissection of 16s rrna methyltransferase (ksga) function in escherichia coli. | a 16s rrna methyltransferase, ksga, identified originally in escherichia coli is highly conserved in all living cells, from bacteria to humans. ksga orthologs in eukaryotes possess functions in addition to their rrna methyltransferase activity. e. coli era is an essential gtp-binding protein. we recently observed that ksga functions as a multicopy suppressor for the cold-sensitive cell growth of an era mutant [era(e200k)] strain (q. lu and m. inouye, j. bacteriol. 180:5243-5246, 1998). here we o ... | 2007 | 17890303 |
| flavin-dependent thymidylate synthase thyx activity: implications for the folate cycle in bacteria. | although flavin-dependent thyx proteins show thymidylate synthase activity in vitro and functionally complement thya defects in heterologous systems, direct proof of their cellular functions is missing. using insertional mutagenesis of rhodobacter capsulatus thyx, we constructed the first defined thyx inactivation mutant. phenotypic analyses of the obtained mutant strain confirmed that r. capsulatus thyx is required for de novo thymidylate synthesis. full complementation of the r. capsulatus thy ... | 2007 | 17890305 |
| deinococcus geothermalis: the pool of extreme radiation resistance genes shrinks. | bacteria of the genus deinococcus are extremely resistant to ionizing radiation (ir), ultraviolet light (uv) and desiccation. the mesophile deinococcus radiodurans was the first member of this group whose genome was completely sequenced. analysis of the genome sequence of d. radiodurans, however, failed to identify unique dna repair systems. to further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second deinococcus species, the thermophile de ... | 2007 | 17895995 |
| functional analysis of the gtpases enga and yhbz encoded by salmonella typhimurium. | the s. typhimurium genome encodes proteins, designated enga and yhbz, which have a high sequence identity with the gtpases enga/der and obge/cgtae of escherichia coli. the wild-type activity of the e. coli proteins is essential for normal ribosome maturation and cell viability. in order to characterize the potential involvement of the salmonella typhimurium enga and yhbz proteins in ribosome biology, we used high stringency affinity chromatography experiments to identify strongly binding ribosom ... | 2007 | 17905831 |
| evaluation of the staphylococcus aureus class c nonspecific acid phosphatase (saps) as a reporter for gene expression and protein secretion in gram-negative and gram-positive bacteria. | a phosphatase secreted by staphylococcus aureus strain 154 has previously been characterized and classified as a new member of the bacterial class c family of nonspecific acid phosphatases. as the acid phosphatase activity can be easily detected with a cost-effective plate screen assay, quantitatively measured by a simple enzyme assay, and detected by zymography, its potential use as a reporter system was investigated. the s. aureus acid phosphatase (saps) gene has been cloned and expressed from ... | 2007 | 17905879 |
| identification of promoters recognized by rna polymerase-sigmae holoenzyme from thermus thermophilus hb8. | thermus thermophilus sigma(e), an extracytoplasmic function sigma factor from the extremely thermophilic bacterium thermus thermophilus hb8, bound to the rna polymerase core enzyme and showed transcriptional activity. with the combination of in vitro transcription assay and genechip technology, we identified three promoters recognized by sigma(e). the predicted consensus promoter sequence for sigma(e) is 5'-ca(a/t)(a/c)c(a/c)-n(15)-ccgta-3'. | 2007 | 17905996 |
| electron transport chain of saccharomyces cerevisiae mitochondria is inhibited by h2o2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement. | the deleterious effects of h202 on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. exposure to h2o2 resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. the effect of h2o2 on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-dcip oxidoreductase activities. inhibitory effect of h2o2 on respiratory complexes was almost completel ... | 2007 | 17907001 |