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dna mismatch repair: molecular mechanism, cancer, and ageing.dna mismatch repair (mmr) proteins are ubiquitous players in a diverse array of important cellular functions. in its role in post-replication repair, mmr safeguards the genome correcting base mispairs arising as a result of replication errors. loss of mmr results in greatly increased rates of spontaneous mutation in organisms ranging from bacteria to humans. mutations in mmr genes cause hereditary nonpolyposis colorectal cancer, and loss of mmr is associated with a significant fraction of sporad ...200818406444
regulation of placental villous angiopoietin-1 and -2 expression by estrogen during baboon pregnancy.we recently showed an increase in vascular endothelial growth factor (vegf), decrease in angiopoietin-1 (ang-1) and unaltered ang-2 expression by the villous placenta with advancing baboon pregnancy. moreover, placental vegf expression was increased by estrogen in early pregnancy. in the present study, we determined whether placental ang-1 and ang-2 are regulated by estrogen. ang-1 and ang-2 mrna and protein were determined by rt-pcr and immunocytochemistry in the placenta of baboons on day 60 o ...200818022824
an intramolecular fret system monitors fingers subdomain opening in klentaq1.a major goal of polymerase research is to determine the mechanism through which a nucleotide complementary to a templating dna base is selected and delivered to the polymerase active site. structural evidence suggests a large open-to-closed conformational change affecting the fingers subdomain as being crucial to the process. we previously designed a fret system capable of measuring the rate of fingers subdomain closure in the presence of correct nucleotide. however, this fret system was limited ...200818287276
"hot cores" in proteins: comparative analysis of the apolar contact area in structures from hyper/thermophilic and mesophilic organisms.a wide variety of stabilizing factors have been invoked so far to elucidate the structural basis of protein thermostability. these include, amongst the others, a higher number of ion-pairs interactions and hydrogen bonds, together with a better packing of hydrophobic residues. it has been frequently observed that packing of hydrophobic side chains is improved in hyperthermophilic proteins, when compared to their mesophilic counterparts. in this work, protein crystal structures from hyper/thermop ...200818312638
simultaneous fitting of real-time pcr data with efficiency of amplification modeled as gaussian function of target fluorescence.in real-time pcr, it is necessary to consider the efficiency of amplification (ea) of amplicons in order to determine initial target levels properly. eas can be deduced from standard curves, but these involve extra effort and cost and may yield invalid eas. alternatively, ea can be extracted from individual fluorescence curves. unfortunately, this is not reliable enough.200818267040
high-throughput avian molecular sexing by sybr green-based real-time pcr combined with melting curve analysis.combination of chd (chromo-helicase-dna binding protein)-specific polymerase chain reaction (pcr) with electrophoresis (pcr/electrophoresis) is the most common avian molecular sexing technique but it is lab-intensive and gel-required. gender determination often fails when the difference in length between the pcr products of chd-z and chd-w genes is too short to be resolved.200818269737
dynamics of recognition between trna and elongation factor tu.elongation factor tu (ef-tu) binds to all standard aminoacyl transfer rnas (aa-trnas) and transports them to the ribosome while protecting the ester linkage between the trna and its cognate amino acid. we use molecular dynamics simulations to investigate the dynamics of the ef-tu.guanosine 5'-triphosphate.aa-trna(cys) complex and the roles played by mg2+ ions and modified nucleosides on the free energy of protein.rna binding. individual modified nucleosides have pronounced effects on the structu ...200818336835
replisome dynamics and use of dna trombone loops to bypass replication blocks.replisomes are dynamic multiprotein machines capable of simultaneously replicating both strands of the dna duplex. this review focuses on the structure and function of the e. coli replisome, many features of which generalize to other bacteria and eukaryotic cells. for example, the bacterial replisome utilizes clamps and clamp loaders to coordinate the actions required of the trombone model of lagging strand synthesis made famous by bruce alberts. all cells contain clamps and clamp loaders and th ...200818931783
ssb as an organizer/mobilizer of genome maintenance complexes.when duplex dna is altered in almost any way (replicated, recombined, or repaired), single strands of dna are usually intermediates, and single-stranded dna binding (ssb) proteins are present. these proteins have often been described as inert, protective dna coatings. continuing research is demonstrating a far more complex role of ssb that includes the organization and/or mobilization of all aspects of dna metabolism. escherichia coli ssb is now known to interact with at least 14 other proteins ...200818937104
a full-length group 1 bacterial sigma factor adopts a compact structure incompatible with dna binding.the sigma factors are the key regulators of bacterial transcription initiation. through direct read-out of promoter dna sequence, they recruit the core rna polymerase to sites of initiation, thereby dictating the rna polymerase promoter-specificity. the group 1 sigma factors, which direct the vast majority of transcription initiation during log phase growth and are essential for viability, are autoregulated by an n-terminal sequence known as sigma1.1. we report the solution structure of thermoto ...200818940669
structures of srp54 and srp19, the two proteins that organize the ribonucleic core of the signal recognition particle from pyrococcus furiosus.in all organisms the signal recognition particle (srp), binds to signal sequences of proteins destined for secretion or membrane insertion as they emerge from translating ribosomes. in archaea and eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein srp19, the essential gtpase srp54, and an evolutionarily conserved and essential srp rna. through the gtp-dependent interaction between the srp and its cognate receptor sr, ribosomes harboring nascent polyp ...200818953414
mismatched base-pair simulations for asfv pol x/dna complexes help interpret frequent g*g misincorporation.dna polymerase x (pol x) from the african swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. surprisingly, pol x's insertion rate of the g*g mispair is comparable to that of the four watson-crick base pairs. this behavior is in contrast with another x-family polymerase, dna polymerase beta (pol beta), which inserts g*g mismatches poorly, and has higher dna repair fidelity. using molecular dyna ...200818955064
the rna polymerase "switch region" is a target for inhibitors.the alpha-pyrone antibiotic myxopyronin (myx) inhibits bacterial rna polymerase (rnap). here, through a combination of genetic, biochemical, and structural approaches, we show that myx interacts with the rnap "switch region"--the hinge that mediates opening and closing of the rnap active center cleft--to prevent interaction of rnap with promoter dna. we define the contacts between myx and rnap and the effects of myx on rnap conformation and propose that myx functions by interfering with opening ...200818957204
interactions between hiv-1 reverse transcriptase and the downstream template strand in stable complexes with primer-template.human immunodeficiency virus type 1 reverse transcriptase (hiv-1 rt) forms stable ternary complexes in which rt is bound tightly at fixed positions on the primer-template (p/t). we have probed downstream interactions between rt and the template strand in the complex containing the incoming dntp (+1 dntp*rt*p/t complex) and in the complex containing the pyrophosphate analog, foscarnet (foscarnet*rt*p/t complex).200818974785
the helicobacter pylori hpyaxii restriction-modification system limits exogenous dna uptake by targeting gtac sites but shows asymmetric conservation of the dna methyltransferase and restriction endonuclease components.the naturally competent organism helicobacter pylori encodes a large number of restriction-modification (r-m) systems that consist of a restriction endonuclease and a dna methyltransferase. r-m systems are not only believed to limit dna exchange among bacteria but may also have other cellular functions. we report a previously uncharacterized h. pylori type ii r-m system, m.hpyaxii/r.hpyaxii. we show that this system targets gtac sites, which are rare in the h. pylori chromosome but numerous in r ...200818978016
structures of the signal recognition particle receptor from the archaeon pyrococcus furiosus: implications for the targeting step at the membrane.in all organisms, a ribonucleoprotein called the signal recognition particle (srp) and its receptor (sr) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. we present the first x-ray structures of an archeal ftsy, the receptor from the hyper-thermophile pyrococcus furiosus (pfu), in its free and gdp*magnesium-bound forms. the highly charged n-terminal domain of pfu-ftsy is distinguished by a long n-terminal helix. the basic charges on the surface of ...200818978942
transposition of an insertion sequence, istth7, in the genome of the extreme thermophile thermus thermophilus hb8.we have identified an active insertion sequence (is) in the genome of thermus thermophilus hb8. transposition was detected as insertional inactivation of a 16s rrna methyltransferase gene, rsmg, resulting in streptomycin resistance. the is element, istth7, is 1029 bp in length, encodes an imperfect 12 bp inverted repeat, and produces a 9 bp direct repeat of the target sequence. the sequence of a putative transposase encoded by istth7 indicates that it is a member of the is427 group within the is ...200819016874
recombinant production and biochemical characterization of a hyperthermostable alpha-glucan/maltodextrin phosphorylase from pyrococcus furiosus.alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-d-glucose-1-phosphate. we report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (pf1535) from a hyperthermophilic archaeon, pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. the apparent 98 kda recombinant enzyme was active over a broad range of temperatures and ...200819054743
recombinant production and biochemical characterization of a hyperthermostable alpha-glucan/maltodextrin phosphorylase from pyrococcus furiosus.alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-d-glucose-1-phosphate. we report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (pf1535) from a hyperthermophilic archaeon, pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. the apparent 98 kda recombinant enzyme was active over a broad range of temperatures and ...200819054743
bridge helix and trigger loop perturbations generate superactive rna polymerases.cellular rna polymerases are highly conserved enzymes that undergo complex conformational changes to coordinate the processing of nucleic acid substrates through the active site. two domains in particular, the bridge helix and the trigger loop, play a key role in this mechanism by adopting different conformations at various stages of the nucleotide addition cycle. the functional relevance of these structural changes has been difficult to assess from the relatively small number of static crystal ...200819055851
genomic sequence and activity of ks10, a transposable phage of the burkholderia cepacia complex.the burkholderia cepacia complex (bcc) is a versatile group of gram negative organisms that can be found throughout the environment in sources such as soil, water, and plants. while bcc bacteria can be involved in beneficial interactions with plants, they are also considered opportunistic pathogens, specifically in patients with cystic fibrosis and chronic granulomatous disease. these organisms also exhibit resistance to many antibiotics, making conventional treatment often unsuccessful. ks10 wa ...200819094239
evidence for involvement of tnfr1 and timps in pathogenesis of post-kala-azar dermal leishmaniasis.semi-quantitative rt-pcr was exploited to analyse the intralesional cytokine gene expression in 14 post-kala-azar dermal leishmaniasis (pkdl) and 10 kala-azar (ka) patients. the data provided evidence for both inflammatory and non-inflammatory responses, as reflected by elevated tumour necrosis factor (tnf)-alpha and interleukin (il)-10 in pkdl lesions compared with normal skin tissue (n = 6). the ratio of tnf-alpha : il-10 message was 2.66 in pkdl cases, substantially higher than in ka (1.18). ...200819037922
structure of polc reveals unique dna binding and fidelity determinants.polc is the polymerase responsible for genome duplication in many gram-positive bacteria and represents an attractive target for antibacterial development. we have determined the 2.4-a resolution crystal structure of geobacillus kaustophilus polc in a ternary complex with dna and dgtp. the structure reveals nascent base pair interactions that lead to highly accurate nucleotide incorporation. a unique beta-strand motif in the polc thumb domain contacts the minor groove, allowing replication error ...200819106298
a comprehensive collection of experimentally validated primers for polymerase chain reaction quantitation of murine transcript abundance.quantitative polymerase chain reaction (qpcr) is a widely applied analytical method for the accurate determination of transcript abundance. primers for qpcr have been designed on a genomic scale but non-specific amplification of non-target genes has frequently been a problem. although several online databases have been created for the storage and retrieval of experimentally validated primers, only a few thousand primer pairs are currently present in existing databases and the primers are not des ...200819108745
evidence for a complex mosaic genome pattern in a full-length hepatitis c virus sequence.the genome of the hepatitis c virus (hcv) exhibits a high genetic variability. this remarkable heterogeneity is mainly attributed to the gradual accumulation of mutational changes, whereas the contribution of recombination events to the evolution of hcv remains controversial so far. while performing phylogenetic analyses including a large number of sequences deposited in the genbank, we encountered a full-length hcv sequence (ay651061) that showed evidence for inter-subtype recombination and was ...200819204822
formation of template-switching artifacts by linear amplification.linear amplification is a method of synthesizing single-stranded dna from either a single-stranded dna or one strand of a double-stranded dna. in this protocol, molecules of a single primer dna are extended by multiple rounds of dna synthesis at high temperature using thermostable dna polymerases. although linear amplification generates the intended full-length single-stranded product, it is more efficient over single-stranded templates than double-stranded templates. we analyzed linear amplific ...200819137105
srp rna controls a conformational switch regulating the srp-srp receptor interaction.the interaction of the signal-recognition particle (srp) with its receptor (sr) mediates co-translational protein targeting to the membrane. srp and sr interact via their homologous core gtpase domains and n-terminal four-helix bundles (n domains). srp-sr complex formation is slow unless catalyzed by srp's essential rna component. we show that truncation of the first helix of the n domain (helix n1) of both proteins dramatically accelerates their interaction. srp and sr with helix n1 truncations ...200819172744
a new family of polymerases related to superfamily a dna polymerases and t7-like dna-dependent rna polymerases.using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain dna viruses, including the recently reported sputnik virus. using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and dna polymerase activity, comparable to the previously reported prim-po ...200818834537
the last universal common ancestor: emergence, constitution and genetic legacy of an elusive forerunner.since the reclassification of all life forms in three domains (archaea, bacteria, eukarya), the identity of their alleged forerunner (last universal common ancestor or luca) has been the subject of extensive controversies: progenote or already complex organism, prokaryote or protoeukaryote, thermophile or mesophile, product of a protracted progression from simple replicators to complex cells or born in the cradle of "catalytically closed" entities? we present a critical survey of the topic and s ...200818613974
expression of placental neurotrophin-3 (nt-3) in physiological pregnancy, preeclampsia and chorioamnionitis.neurotrophic factors are a group of proteins that act as paracrine and autocrine growth factors. they are involved in the regulation of morphogenesis and development of several tissues. the present study aims to evaluate, for the first time, the expression of neurotrophin-3 in the human placenta during normal pregnancy and in preeclampsia and chorioamnionitis. neurotrophin-3 mrna, assessed by rt-pcr analysis in six term placentas, were observed in all the specimens examined. neurotrophin-3 prote ...200921151544
structures of rna polymerase-antibiotic complexes.inhibition of bacterial rna polymerase (rnap) is an established strategy for antituberculosis therapy and broad-spectrum antibacterial therapy. crystal structures of rnap-inhibitor complexes are available for four classes of antibiotics: rifamycins, sorangicin, streptolydigin, and myxopyronin. the structures define three different targets, and three different mechanisms, for inhibition of bacterial rnap: (1) rifamycins and sorangicin bind near the rnap active center and block extension of rna pr ...200919926275
molecular evolution of multisubunit rna polymerases: sequence analysis.transcription in all cellular organisms is performed by multisubunit, dna-dependent rna polymerases that synthesize rna from dna templates. previous sequence and structural studies have elucidated the importance of shared regions common to all multisubunit rna polymerases. in addition, rna polymerases contain multiple lineage-specific domain insertions involved in protein-protein and protein-nucleic acid interactions. we have created comprehensive multiple sequence alignments using all available ...200919895820
molecular evolution of multisubunit rna polymerases: sequence analysis.transcription in all cellular organisms is performed by multisubunit, dna-dependent rna polymerases that synthesize rna from dna templates. previous sequence and structural studies have elucidated the importance of shared regions common to all multisubunit rna polymerases. in addition, rna polymerases contain multiple lineage-specific domain insertions involved in protein-protein and protein-nucleic acid interactions. we have created comprehensive multiple sequence alignments using all available ...200919895820
optical tweezers experiments resolve distinct modes of dna-protein binding.optical tweezers are ideally suited to perform force microscopy experiments that isolate a single biomolecule, which then provides multiple binding sites for ligands. the captured complex may be subjected to a spectrum of forces, inhibiting or facilitating ligand activity. in the following experiments, we utilize optical tweezers to characterize and quantify dna binding of various ligands. high mobility group type b (hmgb) proteins, which bind to double-stranded dna, are shown to serve the dual ...200919173290
correlation of the 4977 bp mitochondrial dna deletion with human sperm dysfunction.several studies have examined the association between mitochondrial dna (mtdna) deletions, in particular the "common" 4977-bp deletion, and human sperm dysfunction, but have produced contradictory results.200919192313
a two-subunit bacterial sigma-factor activates transcription in bacillus subtilis.the sigma-like factor yvri and coregulator yvrha activate transcription from a small set of conserved promoters in bacillus subtilis. we report here that these two proteins independently contribute sigma-region 2 and sigma-region 4 functions to a holoenzyme-promoter dna complex. yvri binds rna polymerase (rnap) through a region 4 interaction with the beta-subunit flap domain and mediates specific promoter recognition but cannot initiate dna melting at the -10 promoter element. conversely, yvrha ...200919940246
rna-protein mutually induced fit: structure of escherichia coli isopentenyl-trna transferase in complex with trna(phe).trnas that read codons starting with u are usually modified at their a37 by isopentenyl-trna transferases to minimize peptidyl-trna slippage in translation. the consensus substrate requirements of the isopentenyl-trna transferase of escherichia coli, miaa, have been the focus of extensive study. however, the molecular basis of trna-miaa recognition remains unknown. here we describe the 2.5a crystal structure of miaa in complex with substrate trna(phe). comparative structural analysis reveals tha ...200919158097
a novel bicistronic vector for overexpressing mycobacterium tuberculosis proteins in escherichia coli.a putative dna glycosylase encoded by the rv3297 gene (mtunei2) has been identified in mycobacterium tuberculosis. our efforts to express this gene in escherichia coli either by supplementing trnas for rare codons or optimizing the gene with preferred codons for e. coli resulted in little or no expression. on the other hand, high-level expression was observed using a bicistronic expression vector in which the target gene was translationally coupled to an upstream leader sequence. further compari ...200919162193
a novel bicistronic vector for overexpressing mycobacterium tuberculosis proteins in escherichia coli.a putative dna glycosylase encoded by the rv3297 gene (mtunei2) has been identified in mycobacterium tuberculosis. our efforts to express this gene in escherichia coli either by supplementing trnas for rare codons or optimizing the gene with preferred codons for e. coli resulted in little or no expression. on the other hand, high-level expression was observed using a bicistronic expression vector in which the target gene was translationally coupled to an upstream leader sequence. further compari ...200919162193
structural basis for catalysis of a tetrameric class iia fructose 1,6-bisphosphate aldolase from mycobacterium tuberculosis.mycobacterium tuberculosis, the causative agent of tuberculosis (tb), currently infects one-third of the world's population in its latent form. the emergence of multidrug-resistant and extensive drug-resistant strains has highlighted the need for new pharmacological targets within m. tuberculosis. the class iia fructose 1,6-bisphosphate aldolase (fba) enzyme from m. tuberculosis (mtfba) has been proposed as one such target since it is upregulated in latent tb. since the structure of mtfba has no ...200919167403
allosteric control of escherichia coli rrna promoter complexes by dksa.the escherichia coli dksa protein inserts into the rna polymerase (rnap) secondary channel, modifying the transcription initiation complex so that promoters with specific kinetic characteristics are regulated by changes in the concentrations of ppgpp and ntps. we used footprinting assays to determine the specific kinetic intermediate, rp(i), on which dksa acts. genetic approaches identified substitutions in the rnap switch regions, bridge helix, and trigger loop that mimicked, reduced, or enhanc ...200919171784
the mechanistic architecture of thermostable pyrococcus furiosus family b dna polymerase motif a and its interaction with the dntp substrate.thermostable dna polymerases isolated from archaeal organisms have not been completely characterized kinetically and require further study if we are to understand both their dntp binding mechanism and their role within the organism. here, we demonstrate that the thermostable family b dna polymerase from pyrococcus furiosus (pfu pol) contains sensitive determinants of both dntp binding and replicational fidelity within the highly conserved motif a. site-directed mutagenesis of the motif a sylp re ...200919817489
three-dimensional structure of n-terminal domain of dnab helicase and helicase-primase interactions in helicobacter pylori.replication initiation is a crucial step in genome duplication and homohexameric dnab helicase plays a central role in the replication initiation process by unwinding the duplex dna and interacting with several other proteins during the process of replication. n-terminal domain of dnab is critical for helicase activity and for dnag primase interactions. we present here the crystal structure of the n-terminal domain (ntd) of h. pylori dnab (hpdnab) helicase at 2.2 a resolution and compare the str ...200919841750
reconstitution of gloeobacter violaceus rhodopsin with a light-harvesting carotenoid antenna.we show that salinixanthin, the light-harvesting carotenoid antenna of xanthorhodopsin, can be reconstituted into the retinal protein from gloeobacter violaceus expressed in escherichia coli. reconstitution of gloeobacter rhodopsin with the carotenoid is accompanied by characteristic absorption changes and the appearance of cd bands similar to those observed for xanthorhodopsin that indicate immobilization and twist of the carotenoid in the binding site. as in xanthorhodopsin, the carotenoid fun ...200919842712
allosteric control of catalysis by the f loop of rna polymerase.bacterial rna polymerases (rnaps) undergo coordinated conformational changes during catalysis. in particular, concerted folding of the trigger loop and rearrangements of the bridge helix at the rnap active center have been implicated in nucleotide addition and rnap translocation. at moderate temperatures, the rate of catalysis by rnap from thermophilic thermus aquaticus is dramatically reduced compared with its closest mesophilic relative, deinococcus radiodurans. here, we show that a part of th ...200919855007
global conformational dynamics of a y-family dna polymerase during catalysis.replicative dna polymerases are stalled by damaged dna while the newly discovered y-family dna polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. the y-family dna polymerases, characterized by low fidelity and processivity, are able to bypass different classes of dna lesions. a variety of kinetic and structural studies have established a minimal reaction pathway common to all dna polymerases, although the conformational intermediates are not wel ...200919859523
cohesion group approach for evolutionary analysis of aspartokinase, an enzyme that feeds a branched network of many biochemical pathways.aspartokinase (ask) exists within a variable network that supports the synthesis of 9 amino acids and a number of other important metabolites. lysine, isoleucine, aromatic amino acids, and dipicolinate may arise from the ask network or from alternative pathways. ask proteins were subjected to cohesion group analysis, a methodology that sorts a given protein assemblage into groups in which evolutionary continuity is assured. two subhomology divisions, ask(alpha) and ask(beta), have been recognize ...200919946135
characterization of dna polymerase x from thermus thermophilus hb8 reveals the polxc and php domains are both required for 3'-5' exonuclease activity.the x-family dna polymerases (polxs) comprise a highly conserved dna polymerase family found in all kingdoms. mammalian polxs are known to be involved in several dna-processing pathways including repair, but the cellular functions of bacterial polxs are less known. many bacterial polxs have a polymerase and histidinol phosphatase (php) domain at their c-termini in addition to a polx core (polxc) domain, and possess 3'-5' exonuclease activity. although both domains are highly conserved in bacteri ...200919211662
formation of multilayered photosynthetic biofilms in an alkaline thermal spring in yellowstone national park, wyoming.in this study, glass rods suspended at the air-water interface in the runoff channel of fairy geyser, yellowstone national park, wy, were used as a substratum to promote the development of biofilms that resembled multilayered mat communities in the splash zone at the geyser's source. this approach enabled the establishment of the temporal relationship between the appearance of cyanobacteria, which ultimately formed the outer green layer, and the development of a red underlayer containing roseifl ...200919218404
stimulation of expression of a silica-induced protein (sip) in thermus thermophilus by supersaturated silicic acid.the effects of silicic acid on the growth of thermus thermophilus tmy, an extreme thermophile isolated from a siliceous deposit formed from geothermal water at a geothermal power plant in japan, were examined at 75 degrees c. at concentrations higher than the solubility of amorphous silica (400 to 700 ppm sio(2)), a silica-induced protein (sip) was isolated from the cell envelope fraction of log-phase tmy cells grown in the presence of supersaturated silicic acid. two-dimensional sodium dodecyl ...200919233950
structural insights into the substrate binding and stereoselectivity of giardia fructose-1,6-bisphosphate aldolase.giardia lamblia fructose-1,6-bisphosphate aldolase (fbpa) is a member of the class ii zinc-dependent aldolase family that catalyzes the cleavage of d-fructose 1,6-bisphosphate (fbp) into dihydroxyacetone phosphate (dhap) and d-glyceraldehyde 3-phosphate (g3p). in addition to the active site zinc, the catalytic apparatus of fbpa employs an aspartic acid, asp83 in the g. lamblia enzyme, which when replaced with an alanine residue renders the enzyme inactive. a comparison of the crystal structures ...200919236002
transcriptional infidelity promotes heritable phenotypic change in a bistable gene network.bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. it is becoming clear that noise in gene expression can influence cell fate. although the origins and consequences of noise have been studied, the stochastic and transient nature of rna errors during transcription has not been considered in the origin or modeling o ...200919243224
deciphering the mismatch recognition cycle in muts and msh2-msh6 using normal-mode analysis.postreplication dna mismatch repair is essential for maintaining the integrity of genomic information in prokaryotes and eukaryotes. the first step in mismatch repair is the recognition of base-base mismatches and insertions/deletions by bacterial muts or eukaryotic msh2-msh6. crystal structures of both proteins bound to mismatch dna reveal a similar molecular architecture but provide limited insight into the detailed molecular mechanism of long-range allostery involved in mismatch recognition a ...200919254532
structural basis for binding of rna and cofactor by a ksga methyltransferase.among methyltransferases, ksga and the reaction it catalyzes are conserved throughout evolution. however, the specifics of substrate recognition by the enzyme remain unknown. here we report structures of aquifex aeolicus ksga, in its ligand-free form, in complex with rna, and in complex with both rna and s-adenosylhomocysteine (sah, reaction product of cofactor s-adenosylmethionine), revealing critical structural information on ksga-rna and ksga-sah interactions. moreover, the structures show ho ...200919278652
the membrane-binding motif of the chloroplast signal recognition particle receptor (cpftsy) regulates gtpase activity.the chloroplast signal recognition particle (cpsrp) and its receptor (cpftsy) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. unlike cytosolic srp receptors in eukaryotes, cpftsy partitions between thylakoid membranes and the soluble stroma. based on sequence alignments, a membrane-binding motif identified in escherichia coli ftsy appears to be conserved in cpftsy, yet whether the proposed motif is responsible for the membrane-binding function of cpftsy has y ...200919293157
mechanism of cadmium-mediated inhibition of msh2-msh6 function in dna mismatch repair.the observation that cadmium (cd(2+)) inhibits msh2-msh6, which is responsible for identifying base pair mismatches and other discrepancies in dna, has led to the proposal that selective targeting of this protein and consequent suppression of dna repair or apoptosis promote the carcinogenic effects of the heavy metal toxin. it has been suggested that cd(2+) binding to specific sites on msh2-msh6 blocks its dna binding and atpase activities. to investigate the mechanism of inhibition, we measured ...200919320425
maintenance of rna-dna hybrid length in bacterial rna polymerases.during transcription elongation the nascent rna remains base-paired to the template strand of the dna before it is displaced and the two strands of the dna reanneal, resulting in the formation of a transcription "bubble" of approximately 10 bp. to examine how the length of the rna-dna hybrid is maintained, we assembled transcription elongation complexes on synthetic nucleic acid scaffolds that mimic the situation in which transcript displacement is compromised and the polymerase synthesizes an e ...200919321439
effect of n-2-acetylaminofluorene and 2-aminofluorene adducts on dna binding and synthesis by yeast dna polymerase eta.the well-studied aromatic amine carcinogen, n-2-acetylaminofluorene (aaf), forms adducts at the c8 position of guanine in dna. unlike replicative polymerases, y-family polymerases have been shown to have the ability to bypass such bulky dna lesions. to better understand the mechanism of translesion synthesis by the yeast dna polymerase eta (ypoleta), a gel retardation technique was used to measure equilibrium dissociation constants of this polymerase for unmodified dna or dna containing dg-c8-aa ...200919354292
qualitative and quantitative detection of chlamydophila pneumoniae dna in cerebrospinal fluid from multiple sclerosis patients and controls.a standardized molecular test for the detection of chlamydophila pneumoniae dna in cerebrospinal fluid (csf) would assist the further assessment of the association of c. pneumoniae with multiple sclerosis (ms). we developed and validated a qualitative colorimetric microtiter plate-based pcr assay (pcr-eia) and a real-time quantitative pcr assay (taqman) for detection of c. pneumoniae dna in csf specimens from ms patients and controls. compared to a touchdown nested-pcr assay, the sensitivity, sp ...200919357786
low-fidelity dna synthesis by the l979f mutator derivative of saccharomyces cerevisiae dna polymerase zeta.to probe pol zeta functions in vivo via its error signature, here we report the properties of saccharomyces cerevisiae pol zeta in which phenyalanine was substituted for the conserved leu-979 in the catalytic (rev3) subunit. we show that purified l979f pol zeta is 30% as active as wild-type pol zeta when replicating undamaged dna. l979f pol zeta shares with wild-type pol zeta the ability to perform moderately processive dna synthesis. when copying undamaged dna, l979f pol zeta is error-prone com ...200919380376
mutational analysis of escherichia coli sigma28 and its target promoters reveals recognition of a composite -10 region, comprised of an 'extended -10' motif and a core -10 element.sigma28 controls the expression of flagella-related genes and is the most widely distributed alternative sigma factor, present in motile gram-positive and gram-negative bacteria. the distinguishing feature of sigma28 promoters is a long -10 region (gccgataa). despite the fact that the upstream gc is highly conserved, previous studies have not indicated a functional role for this motif. here we examine the functional relevance of the gccg motif and determine which residues in sigma28 participate ...200919400790
dissection of recognition determinants of escherichia coli sigma32 suggests a composite -10 region with an 'extended -10' motif and a core -10 element.sigma32 controls expression of heat shock genes in escherichia coli and is widely distributed in proteobacteria. the distinguishing feature of sigma32 promoters is a long -10 region (ccccatnt) whose tetra-c motif is important for promoter activity. using alanine-scanning mutagenesis of sigma32 and in vivo and in vitro assays, we identified promoter recognition determinants of this motif. the most downstream c (-13) is part of the -10 motif; our work confirms and extends recognition determinants ...200919400791
the optimization of taqman real-time rt-pcr assay for transcriptional profiling of gaba-a receptor subunit plasticity.the gaba-a receptor plays a critical role in inhibitory neurotransmission in the brain. quantitation of gaba-a receptor subunits in various brain regions is essential to understand their role in plasticity and brain disorders. however, conventional rna assays are tedious and less sensitive for use in studies of subunit plasticity. here we describe optimization of a sensitive assay of gaba-a receptor subunit gene expression by taqman real-time pcr. for each subunit gene, a set of primers and taqm ...200919406150
structure, stability, and folding of ribonuclease h1 from the moderately thermophilic chlorobium tepidum: comparison with thermophilic and mesophilic homologues.proteins from thermophilic organisms are able to function under conditions that render a typical mesophilic protein inactive. pairwise comparisons of homologous mesophilic and thermophilic proteins can help to identify the energetic features of a protein's energy landscape that lead to such thermostability. previous studies of bacterial ribonucleases h (rnases h) from the thermophile thermus thermophilus and the mesophile escherichia coli revealed that the thermostability arises in part from an ...200919408959
evolution of complex rna polymerases: the complete archaeal rna polymerase structure.the archaeal rna polymerase (rnap) shares structural similarities with eukaryotic rnap ii but requires a reduced subset of general transcription factors for promoter-dependent initiation. to deepen our knowledge of cellular transcription, we have determined the structure of the 13-subunit dna-directed rnap from sulfolobus shibatae at 3.35 a resolution. the structure contains the full complement of subunits, including rpog/rpb8 and the equivalent of the clamp-head and jaw domains of the eukaryoti ...200919419240
assay and purification of omega-amidase/nit2, a ubiquitously expressed putative tumor suppressor, that catalyzes the deamidation of the alpha-keto acid analogues of glutamine and asparagine.omega-amidase (omega-amidodicarboxylate amidohydrolase, ec 3.5.1.3) isolated from rat liver cytosol is a versatile enzyme that catalyzes a large number of amidase, transamidase, and ester hydrolysis reactions. omega-amidase activity toward alpha-ketoglutaramate and alpha-ketosuccinamate (the alpha-keto acid analogues of glutamine and asparagine, respectively) is present in mammalian tissues, tumors, plants, bacteria, and fungi. despite its versatility, widespread occurrence, and high specific ac ...200919464248
structure of the human rev1-dna-dntp ternary complex.y-family dna polymerases have proven to be remarkably diverse in their functions and in strategies for replicating through dna lesions. the structure of yeast rev1 ternary complex has revealed the most radical replication strategy, where the polymerase itself dictates the identity of the incoming nucleotide, as well as the identity of the templating base. we show here that many of the key elements of this highly unusual strategy are conserved between yeast and human rev1, including the eviction ...200919464298
comprehensive analysis of phosphorylated proteins of escherichia coli ribosomes.phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. in this study, we have mapped the specific phosphorylation sites in 24 escherichia coli ribosomal proteins by tandem mass spectrometry. detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, pr ...200919469554
developmental validation of short tandem repeat reagent kit for forensic dna profiling of canine biological material.to develop a reagent kit that enables multiplex polymerase chain reaction (pcr) amplification of 18 short tandem repeats (str) and the canine sex-determining zinc finger marker.200919480022
cloning and analysis of a bifunctional methyltransferase/restriction endonuclease tspgwi, the prototype of a thermus sp. enzyme family.restriction-modification systems are a diverse class of enzymes. they are classified into four major types: i, ii, iii and iv. we have previously proposed the existence of a thermus sp. enzyme family, which belongs to type ii restriction endonucleases (reases), however, it features also some characteristics of types i and iii. members include related thermophilic endonucleases: tspgwi, taqii, tspdti, and tth111ii.200919480701
neuroprotective effects of the triterpenoid, cddo methyl amide, a potent inducer of nrf2-mediated transcription.the nf-e2-related factor-2 (nrf2)/antioxidant response element (are) signaling pathway regulates phase 2 detoxification genes, including a variety of antioxidative enzymes. we tested neuroprotective effects of the synthetic triterpenoid cddo-ma, a potent activator of the nrf2/are signaling. cddo-ma treatment of neuroblastoma sh-sy5y cells resulted in nrf2 upregulation and translocation from cytosol to nucleus and subsequent activation of are pathway genes. cddo-ma blocked t-butylhydroperoxide-in ...200919484125
at the crossroads of bacterial metabolism and virulence factor synthesis in staphylococci.bacteria live in environments that are subject to rapid changes in the availability of the nutrients that are necessary to provide energy and biosynthetic intermediates for the synthesis of macromolecules. consequently, bacterial survival depends on the ability of bacteria to regulate the expression of genes coding for enzymes required for growth in the altered environment. in pathogenic bacteria, adaptation to an altered environment often includes activating the transcription of virulence genes ...200919487727
structure of human dna polymerase kappa inserting datp opposite an 8-oxog dna lesion.oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in dna and 7,8-dihydro-8-oxoguanine (8-oxog) is one of the major lesions formed. it is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxog(syn) can pair with an a in addition to normal base pairing of 8-oxog(anti) with a c. human dna polymerase kappa (polkappa) is a member of the newly discovered y-family of dna polymerases that possess the ability to replicate through ...200919492058
engineering a leucine zipper-trail homotrimer with improved cytotoxicity in tumor cells.successful cancer therapies aim to induce selective apoptosis in neoplastic cells. the current suboptimal efficiency and selectivity drugs have therapeutic limitations and induce concomitant side effects. recently, novel cancer therapies based on the use of tumor necrosis factor-related apoptosis-inducing ligand (trail) have emerged. trail, a key component of the natural antitumor immune response, selectively kills many tumor cell types. earlier studies with recombinant trail, however, revealed ...200919509255
plasmid-based laczalpha assay for dna polymerase fidelity: application to archaeal family-b dna polymerase.the preparation of a gapped puc18 derivative, containing the laczalpha reporter gene in the single-stranded region, is described. gapping is achieved by flanking the laczalpha gene with sites for two related nicking endonucleases, enabling the excision of either the coding or non-coding strand. however, the excised strand remains annealed to the plasmid through non-covalent watson-crick base-pairing; its removal, therefore, requires a heat-cool cycle in the presence of an exactly complementary c ...200919515939
6s rna binding to esigma(70) requires a positively charged surface of sigma(70) region 4.2.6s rna is a small, non-coding rna that interacts with sigma(70)-rna polymerase and downregulates transcription at many promoters during stationary phase. when bound to sigma(70)-rna polymerase, 6s rna is engaged in the active site of sigma(70)-rna polymerase in a manner similar enough to promoter dna that the rna can serve as a template for rna synthesis. it has been proposed that 6s rna mimics the conformation of dna during transcription initiation, suggesting contacts between rna polymerase an ...200919538447
the closest relatives of icosahedral viruses of thermophilic bacteria are among viruses and plasmids of the halophilic archaea.we have sequenced the genome and identified the structural proteins and lipids of the novel membrane-containing, icosahedral virus p23-77 of thermus thermophilus. p23-77 has an approximately 17-kb circular double-stranded dna genome, which was annotated to contain 37 putative genes. virions were subjected to dissociation analysis, and five protein species were shown to associate with the internal viral membrane, while three were constituents of the protein capsid. analysis of the bacteriophage g ...200919587059
a structural view on the mechanism of the ribosome-catalyzed peptide bond formation.the ribosome is a large ribonucleoprotein particle that translates genetic information encoded in mrna into specific proteins. its highly conserved active site, the peptidyl-transferase center (ptc), is located on the large (50s) ribosomal subunit and is comprised solely of rrna, which makes the ribosome the only natural ribozyme with polymerase activity. the last decade witnessed a rapid accumulation of atomic-resolution structural data on both ribosomal subunits as well as on the entire riboso ...200919595805
cell surface engineering of a beta-galactosidase for galactooligosaccharide synthesis.a novel gene encoding transglycosylating beta-galactosidase (bgase) was cloned from penicillium expansum f3. the sequence contained a 3,036-bp open reading frame encoding a 1,011-amino-acid protein. this gene was subsequently expressed on the cell surface of saccharomyces cerevisiae eby-100 by galactose induction. the bgase-anchored yeast could directly utilize lactose to produce galactooligosaccharide (gos), as well as the by-products glucose and a small quantity of galactose. the glucose was c ...200919617384
interaction of human dna polymerase alpha and dna polymerase i from bacillus stearothermophilus with hypoxanthine and 8-oxoguanine nucleotides.to better understand how dna polymerases interact with mutagenic bases, we examined how human dna polymerase alpha (pol alpha), a b family enzyme, and dna polymerase from bacillus stearothermophilus (bf), an a family enzyme, generate adenine:hypoxanthine and adenine:8-oxo-7,8-dihydroguanine (8-oxog) base pairs. pol alpha strongly discriminated against polymerizing datp opposite 8-oxog, and removing n1, n(6), or n7 further inhibited incorporation, whereas removing n3 from datp dramatically increa ...200919642651
impaired mobilization of hematopoietic stem/progenitor cells in c5-deficient mice supports the pivotal involvement of innate immunity in this process and reveals novel promobilization effects of granulocytes.we reported that complement cascade (cc) becomes activated in bone marrow (bm) during granulocyte colony-stimulating factor (g-csf) mobilization of hematopoietic stem/progenitor cells (hspcs) and showed that, although third cc component (c3)-deficient mice are easy mobilizers, fifth cc component (c5)-deficient mice mobilize very poorly. to explain this, we postulated that activation/cleavage of cc releases c3a and c5a anaphylatoxins that differently regulate mobilization. accordingly, c3a, by en ...200919657368
complexity of the msg gene family of pneumocystis carinii.the relationship between the parasitic fungus pneumocystis carinii and its host, the laboratory rat, presumably involves features that allow the fungus to circumvent attacks by the immune system. it is hypothesized that the major surface glycoprotein (msg) gene family endows pneumocystis with the capacity to vary its surface. this gene family is comprised of approximately 80 genes, which each are approximately 3 kb long. expression of the msg gene family is regulated by a cis-dependent mechanism ...200919664205
the structure of bacterial rna polymerase in complex with the essential transcription elongation factor nusa.there are three stages of transcribing dna into rna. these stages are initiation, elongation and termination, and they are well-understood biochemically. however, despite the plethora of structural information made available on rna polymerase in the last decade, little is available for rna polymerase in complex with transcription elongation factors. to understand the mechanisms of transcriptional regulation, we describe the first structure, to our knowledge, for a bacterial rna polymerase in com ...200919680289
crystal structure of human selenocysteine trna.selenocysteine (sec) is the 21st amino acid in translation. sec trna (trna(sec)) has an anticodon complementary to the uga codon. we solved the crystal structure of human trna(sec). trna(sec) has a 9-bp acceptor stem and a 4-bp t stem, in contrast with the 7-bp acceptor stem and the 5-bp t stem in the canonical trnas. the acceptor stem is kinked between the u6:u67 and g7:c66 base pairs, leading to a bent acceptor-t stem helix. trna(sec) has a 6-bp d stem and a 4-nt d loop. the long d stem includ ...200919692584
mechanism of polymerase collision release from sliding clamps on the lagging strand.replicative polymerases are tethered to dna by sliding clamps for processive dna synthesis. despite attachment to a sliding clamp, the polymerase on the lagging strand must cycle on and off dna for each okazaki fragment. in the 'collision release' model, the lagging strand polymerase collides with the 5' terminus of an earlier completed fragment, which triggers it to release from dna and from the clamp. this report examines the mechanism of collision release by the escherichia coli pol iii polym ...200919696739
phosphorylated proteins of the mammalian mitochondrial ribosome: implications in protein synthesis.mitochondria, the powerhouse of eukaryotic cells, have their own translation machinery that is solely responsible for synthesis of 13 mitochondrially encoded protein subunits of oxidative phosphorylation complexes. phosphorylation is a well-known post-translational modification in regulation of many processes in mammalian mitochondria including oxidative phosphorylation. however, there is still very limited knowledge on phosphorylation of mitochondrial ribosomal proteins and their role(s) in rib ...200919702336
the thermus thermophilus dead box helicase hera contains a modified rna recognition motif domain loosely connected to the helicase core.dead box family helicases consist of a helicase core that is formed by two flexibly linked reca-like domains. the helicase activity can be regulated by n- or c-terminal extensions flanking the core. thermus thermophilus heat resistant rna-dependent atpase (hera) is the first dead box helicase that forms a dimer using a unique dimerization domain. in addition to the dimerization domain, hera contains a c-terminal rna binding domain (rbd) that shares sequence homology only to uncharacterized prote ...200919710183
novel human interleukin-15 agonists.il-15 is an immunostimulatory cytokine trans-presented with the il-15 receptor alpha-chain to the shared il-2/il-15rbeta and common gamma-chains displayed on the surface of t cells and nk cells. to further define the functionally important regions of this cytokine, activity and binding studies were conducted on human il-15 muteins generated by site-directed mutagenesis. amino acid substitutions of the asparagine residue at position 72, which is located at the end of helix c, were found to provid ...200919710453
the interaction of bacillus subtilis sigmaa with rna polymerase.rna polymerase (rnap) is an essential and highly conserved enzyme in all organisms. the process of transcription initiation is fundamentally different between prokaryotes and eukaryotes. in prokaryotes, initiation is regulated by sigma factors, making the essential interaction between sigma factors and rnap an attractive target for antimicrobial agents. our objective was to achieve the first step in the process of developing novel antimicrobial agents, namely to prove experimentally that the int ...200919735077
the staphylococcus aureus psk41 plasmid-encoded arta protein is a master regulator of plasmid transmission genes and contains a rhh motif used in alternate dna-binding modes.plasmids harbored by staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. here, we demonstrate that the arta protein encoded by the s. aureus multi-resistance plasmid psk41 is a global transcriptional regulator of psk41 genes, including those involved in conjugation and segregation. arta shows no sequence homology to any structurally characterized dna-b ...200919759211
a trimeric dna polymerase complex increases the native replication processivity.dna polymerases are essential enzymes in all domains of life for both dna replication and repair. the primary dna replication polymerase from sulfolobus solfataricus (ssodpo1) has been shown previously to provide the necessary polymerization speed and exonuclease activity to replicate the genome accurately. we find that this polymerase is able to physically associate with itself to form a trimer and that this complex is stabilized in the presence of dna. analytical gel filtration and electrophor ...200919773426
adenosine triphosphate stimulates aquifex aeolicus mutl endonuclease activity.background: human pms2 (hpms2) homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack muth. mn(++) was previously found to stimulate the endonuclease activity of these homologues. atp was required for the nicking activity of hpms2 and ypms1, but was reported to inhibit bacterial mutl proteins from thermus thermophilus and aquifex aeolicus that displayed homology to hpms2. mutational analysis has identified the dqha(x)(2)e(x)(4)e motif presen ...200919777055
evolving a polymerase for hydrophobic base analogues.hydrophobic base analogues (hbas) have shown great promise for the expansion of the chemical and coding potential of nucleic acids but are generally poor polymerase substrates. while extensive synthetic efforts have yielded examples of hbas with favorable substrate properties, their discovery has remained challenging. here we describe a complementary strategy for improving hba substrate properties by directed evolution of a dedicated polymerase using compartmentalized self-replication (csr) with ...200919778048
dihydroorotase from the hyperthermophile aquifex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis.in prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (cps), aspartate transcarbamoylase (atc), and dihydroorotase (dho), are commonly expressed separately and either function independently (escherichia coli) or associate into multifunctional complexes (aquifex aeolicus). in mammals the enzymes are expressed as a single polypeptide chain (cad) in the order cps-dho-atc and associate into a hexamer. this study presents the three-dimensional structure of ...200919128030
jc virus vp1 loop-specific polymorphisms are associated with favorable prognosis for progressive multifocal leukoencephalopathy.jc virus (jcv) is a human polyomavirus that causes progressive multifocal leukoencephalopathy (pml), a fatal demyelinating disease that mainly affects immunocompromised subjects. since its discovery, pml has been considered a rapidly progressing fatal disease; however, amino acid substitutions in the capsid viral protein have recently been tentatively associated with changes in pml clinical course. in order to provide more insight to pml pathogenesis and identify potential prognostic markers, se ...200919043822
jc virus vp1 loop-specific polymorphisms are associated with favorable prognosis for progressive multifocal leukoencephalopathy.jc virus (jcv) is a human polyomavirus that causes progressive multifocal leukoencephalopathy (pml), a fatal demyelinating disease that mainly affects immunocompromised subjects. since its discovery, pml has been considered a rapidly progressing fatal disease; however, amino acid substitutions in the capsid viral protein have recently been tentatively associated with changes in pml clinical course. in order to provide more insight to pml pathogenesis and identify potential prognostic markers, se ...200919043822
common variants in the nlrp3 region contribute to crohn's disease susceptibility.we used a candidate gene approach to identify a set of snps, located in a predicted regulatory region on chromosome 1q44 downstream of nlrp3 (previously known as cias1 and nalp3) that are associated with crohn's disease. the associations were consistently replicated in four sample sets from individuals of european descent. in the combined analysis of all samples (710 father-mother-child trios, 239 cases and 107 controls), these snps were strongly associated with risk of crohn's disease (p(combin ...200919098911
common variants in the nlrp3 region contribute to crohn's disease susceptibility.we used a candidate gene approach to identify a set of snps, located in a predicted regulatory region on chromosome 1q44 downstream of nlrp3 (previously known as cias1 and nalp3) that are associated with crohn's disease. the associations were consistently replicated in four sample sets from individuals of european descent. in the combined analysis of all samples (710 father-mother-child trios, 239 cases and 107 controls), these snps were strongly associated with risk of crohn's disease (p(combin ...200919098911
conformation of the signal recognition particle in ribosomal targeting complexes.the bacterial signal recognition particle (srp) binds to ribosomes synthesizing inner membrane proteins and, by interaction with the srp receptor, ftsy, targets them to the translocon at the membrane. here we probe the conformation of srp and srp protein, ffh, at different stages of targeting by measuring fluorescence resonance energy transfer (fret) between fluorophores placed at various positions within srp. distances derived from fret indicate that srp binding to nontranslating ribosomes trig ...200919029307
active site substitutions delineate distinct classes of eubacterial flap endonuclease.fens (flap endonucleases) play essential roles in dna replication, pivotally in the resolution of okazaki fragments. in eubacteria, dna poli (polymerase i) contains a flap processing domain, the n-terminal 5'-->3' exonuclease. we present evidence of paralogous fen-encoding genes present in many eubacteria. two distinct classes of these independent fen-encoding genes exist with four groups of eubacteria, being identified based on the number and type of fen gene encoded. the respective proteins po ...200919000038
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