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ubiquitin-like proteins and their roles in archaea.this review highlights the finding that ubiquitin-like (ubl) proteins of archaea (termed samps) function not only as sulfur carriers but also as protein modifiers. ubaa (an e1 ubiquitin-activating enzyme homolog of archaea) is required for the samps to be covalently attached to proteins. the samps and ubaa are also needed to form sulfur-containing biomolecules (e.g., thiolated trna and molybdenum cofactor). these findings provide a new perspective on how ubl proteins can serve as both sulfur car ...201223140889
ubiquitin-like proteins and their roles in archaea.this review highlights the finding that ubiquitin-like (ubl) proteins of archaea (termed samps) function not only as sulfur carriers but also as protein modifiers. ubaa (an e1 ubiquitin-activating enzyme homolog of archaea) is required for the samps to be covalently attached to proteins. the samps and ubaa are also needed to form sulfur-containing biomolecules (e.g., thiolated trna and molybdenum cofactor). these findings provide a new perspective on how ubl proteins can serve as both sulfur car ...201223140889
proteomic properties reveal phyloecological clusters of archaea.in this study, we propose a novel way to describe the variety of environmental adaptations of archaea. we have clustered 57 archaea by using a non-redundant set of proteomic features, and verified that the clusters correspond to environmental adaptations to the archaeal habitats. the first cluster consists dominantly of hyperthermophiles and hyperthermoacidophilic aerobes. the second cluster joins together halophilic and extremely halophilic archaea, while the third cluster contains mesophilic ( ...201223133575
role of the -pewy-glutamate in catalysis at the q(o)-site of the cyt bc(1) complex.we re-examine the ph dependence of partial processes of ubihydroquinone (qh(2)) turnover in glu-295 mutants in rhodobacter sphaeroides to clarify the mechanistic role. in more crippled mutants, the bell-shaped ph profile of wildtype was replaced by dependence on a single pk at ~8.5 favoring electron transfer. loss of the pk at 6.5 reflects a change in the rate-limiting step from the first to the second electron transfer. over the range of ph 6-8, no major ph dependence of formation of the initia ...201223123515
role of the -pewy-glutamate in catalysis at the q(o)-site of the cyt bc(1) complex.we re-examine the ph dependence of partial processes of ubihydroquinone (qh(2)) turnover in glu-295 mutants in rhodobacter sphaeroides to clarify the mechanistic role. in more crippled mutants, the bell-shaped ph profile of wildtype was replaced by dependence on a single pk at ~8.5 favoring electron transfer. loss of the pk at 6.5 reflects a change in the rate-limiting step from the first to the second electron transfer. over the range of ph 6-8, no major ph dependence of formation of the initia ...201223123515
structural asymmetry in the thermus thermophilus ruvc dimer suggests a basis for sequential strand cleavages during holliday junction resolution.holliday junction (hj) resolvases are structure-specific endonucleases that cleave four-way dna junctions (hjs) generated during dna recombination and repair. bacterial ruvc, a prototypical hj resolvase, functions as homodimer and nicks dna strands precisely across the junction point. to gain insights into the mechanisms underlying symmetrical strand cleavages by ruvc, we performed crystallographic and biochemical analyses of ruvc from thermus thermophilus (t.th. ruvc). the crystal structure of ...201223118486
structural asymmetry in the thermus thermophilus ruvc dimer suggests a basis for sequential strand cleavages during holliday junction resolution.holliday junction (hj) resolvases are structure-specific endonucleases that cleave four-way dna junctions (hjs) generated during dna recombination and repair. bacterial ruvc, a prototypical hj resolvase, functions as homodimer and nicks dna strands precisely across the junction point. to gain insights into the mechanisms underlying symmetrical strand cleavages by ruvc, we performed crystallographic and biochemical analyses of ruvc from thermus thermophilus (t.th. ruvc). the crystal structure of ...201223118486
characterization of recombinant fluoroquinolone-resistant pneumococcus-like isolates.fourteen fluoroquinolone-resistant streptococcal isolates with recombinant dna topoisomerase genes, preliminarily identified as pneumococci, were further characterized using phenotypic and genotypic approaches. phenotypic tests classified them as atypical pneumococci. phylogenetic relationships were analyzed by using the sequences of seven housekeeping alleles from these isolates and from isolates of streptococcus pneumoniae, streptococcus mitis, streptococcus oralis, and streptococcus pseudopne ...201323114769
alkyltransferase-like protein (atl1) distinguishes alkylated guanines for dna repair using cation-π interactions.alkyltransferase-like (atl) proteins in schizosaccharomyces pombe (atl1) and thermus thermophilus (ttha1564) protect against the adverse effects of dna alkylation damage by flagging o(6)-alkylguanine lesions for nucleotide excision repair (ner). we show that both atl proteins bind with high affinity to oligodeoxyribonucleotides containing o(6)-alkylguanines differing in size, polarity, and charge of the alkyl group. however, atl1 shows a greater ability than ttha1564 to distinguish between o(6)- ...201223112169
the fungal α-aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerization of homocitrate to homoisocitrate.fungi produce α-aminoadipate, a precursor for penicillin and lysine via the α-aminoadipate pathway. despite the biotechnological importance of this pathway, the essential isomerization of homocitrate via homoaconitate to homoisocitrate has hardly been studied. therefore, we analysed the role of homoaconitases and aconitases in this isomerization. although we confirmed an essential contribution of homoaconitases from saccharomyces cerevisiae and aspergillus fumigatus, these enzymes only catalysed ...201223106124
roles of subunit nuok (nd4l) in the energy-transducing mechanism of escherichia coli ndh-1 (nadh:quinone oxidoreductase).the bacterial h(+)-translocating nadh:quinone oxidoreductase (ndh-1) catalyzes electron transfer from nadh to quinone coupled with proton pumping across the cytoplasmic membrane. the nuok subunit (counterpart of the mitochondrial nd4l subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (tm1-3). two glutamic residues located in the adjacent transmembrane helices of nuok are important for the energy coupled activity of ndh-1. in particula ...201223105119
trmt61b is a methyltransferase responsible for 1-methyladenosine at position 58 of human mitochondrial trnas.in human mitochondria, 1-methyladenosine (m¹a) occurs at position 58 of trna(leu(uur)). in addition, partial m¹a58 modifications have been found in human mitochondrial trna(lys) and trna(ser(ucn)). we identified human trmt61b, which encodes a mitochondria-specific trna methyltransferase responsible for m¹a58 in these three trnas. trmt61b is dominantly localized to the mitochondria. m¹a58 formation in human mitochondrial trna(leu(uur)) could be reconstituted in vitro using recombinant trmt61b in ...201223097428
the trna recognition mechanism of folate/fad-dependent trna methyltransferase (trmfo).the conserved u54 in trna is often modified to 5-methyluridine (m(5)u) and forms a reverse hoogsteen base pair with a58 that stabilizes the l-shaped trna structure. in gram-positive and some gram-negative eubacteria, m(5)u54 is produced by folate/fad-dependent trna (m(5)u54) methyltransferase (trmfo). trmfo utilizes n(5),n(10)-methylenetetrahydrofolate (ch(2)thf) as a methyl donor. we previously reported an in vitro trmfo assay system, in which unstable [(14)c]ch(2)thf was supplied from [(14)c]s ...201223095745
leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.gene expression analysis in leishmania donovani (ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (ldass), that was more abundantly expressed in amastigotes than in promastigotes. in order to characterize in detail this newly identified protein in leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo.201223094117
characterization and structure of the aquifex aeolicus protein duf752: a bacterial trna-methyltransferase (mnmc2) functioning without the usually fused oxidase domain (mnmc1).post-transcriptional modifications of the wobble uridine (u34) of trnas play a critical role in reading nna/g codons belonging to split codon boxes. in a subset of escherichia coli trna, this wobble uridine is modified to 5-methylaminomethyluridine (mnm(5)u34) through sequential enzymatic reactions. uridine 34 is first converted to 5-carboxymethylaminomethyluridine (cmnm(5)u34) by the mnme-mnmg enzyme complex. the cmnm(5)u34 is further modified to mnm(5)u by the bifunctional mnmc protein. in the ...201223091054
redundancy and modularity in membrane-associated dissimilatory nitrate reduction in bacillus.the genomes of two phenotypically denitrifying type strains of the genus bacillus were sequenced and the pathways for dissimilatory nitrate reduction were reconstructed. results suggest that denitrification proceeds in the periplasmic space and in an analogous fashion as in gram-negative organisms, yet with the participation of proteins that tend to be membrane-bound or membrane-associated. a considerable degree of functional redundancy was observed with marked differences between b. azotoforman ...201223087684
the mercury resistance operon: from an origin in a geothermal environment to an efficient detoxification machine.mercuric mercury (hg[ii]) is a highly toxic and mobile element that is likely to have had a pronounced and adverse effect on biology since earth's oxygenation ∼2.4 billion years ago due to its high affinity for protein sulfhydryl groups, which upon binding destabilize protein structure and decrease enzyme activity, resulting in a decreased organismal fitness. the central enzyme in the microbial mercury detoxification system is the mercuric reductase (mera) protein, which catalyzes the reduction ...201223087676
rna polymerase-promoter interactions determining different stability of the escherichia coli and thermus aquaticus transcription initiation complexes.transcription initiation complexes formed by bacterial rna polymerases (rnaps) exhibit dramatic species-specific differences in stability, leading to different strategies of transcription regulation. the molecular basis for this diversity is unclear. promoter complexes formed by rnap from thermus aquaticus (taq) are considerably less stable than escherichia coli rnap promoter complexes, particularly at temperatures below 37°c. here, we used a fluorometric rnap molecular beacon assay to discern p ...201223087380
structural basis of transcription initiation.during transcription initiation, rna polymerase (rnap) binds and unwinds promoter dna to form an rnap-promoter open complex. we have determined crystal structures at 2.9 and 3.0 å resolution of functional transcription initiation complexes comprising thermus thermophilus rna polymerase, σ(a), and a promoter dna fragment corresponding to the transcription bubble and downstream double-stranded dna of the rnap-promoter open complex. the structures show that σ recognizes the -10 element and discrimi ...201223086998
cell-free protein synthesis: the state of the art.cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. like pcr, which uses cellular replication machinery to create a dna amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. by breaking free from the constraints of cell-based systems, it takes the nex ...201223086573
cell-free protein synthesis: the state of the art.cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. like pcr, which uses cellular replication machinery to create a dna amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. by breaking free from the constraints of cell-based systems, it takes the nex ...201223086573
identification and characterization of a highly conserved crenarchaeal protein lysine methyltransferase with broad substrate specificity.protein lysine methylation occurs extensively in the crenarchaeota, a major kingdom in the archaea. however, the enzymes responsible for this type of posttranslational modification have not been found. here we report the identification and characterization of the first crenarchaeal protein lysine methyltransferase, designated akmt, from the hyperthermophilic crenarchaeon sulfolobus islandicus. the enzyme was capable of transferring methyl groups to selected lysine residues in a substrate protein ...201223086207
interplay of dna repair with transcription: from structures to mechanisms.many dna transactions are crucial for maintaining genomic integrity and faithful transfer of genetic information but remain poorly understood. an example is the interplay between nucleotide excision repair (ner) and transcription, also known as transcription-coupled dna repair (tcr). discovered decades ago, the mechanisms for tcr have remained elusive, not in small part due to the scarcity of structural studies of key players. here we summarize recent structural information on ner/tcr factors, f ...201223084398
nucleic acid binding surface and dimer interface revealed by crispr-associated casb protein structures.the crispr system is an adaptive rna-based microbial immune system against invasive genetic elements. casb is an essential protein component in type i-e cascade. here, we characterize casb proteins from three different organisms as non-specific nucleic acid binding proteins. the thermobifida fusca casb crystal structure reveals conserved positive surface charges, which we show are important for its nucleic acid binding function. em docking reveals that casb dimerization aligns individual nucleic ...201223079036
recent advances in the structural mechanisms of dna glycosylases.dna glycosylases safeguard the genome by locating and excising a diverse array of aberrant nucleobases created from oxidation, alkylation, and deamination of dna. since the discovery 28years ago that these enzymes employ a base flipping mechanism to trap their substrates, six different protein architectures have been identified to perform the same basic task. work over the past several years has unraveled details for how the various dna glycosylases survey dna, detect damage within the duplex, s ...201223076011
recent advances in the structural mechanisms of dna glycosylases.dna glycosylases safeguard the genome by locating and excising a diverse array of aberrant nucleobases created from oxidation, alkylation, and deamination of dna. since the discovery 28years ago that these enzymes employ a base flipping mechanism to trap their substrates, six different protein architectures have been identified to perform the same basic task. work over the past several years has unraveled details for how the various dna glycosylases survey dna, detect damage within the duplex, s ...201223076011
a mutation of the rna polymerase β' subunit (rpoc) confers cephalosporin resistance in bacillus subtilis.in bacteria, mutations affecting the major catalytic subunits of rna polymerase (encoded by rpob and rpoc) emerge in response to a variety of selective pressures. here we isolated a bacillus subtilis strain with high-level resistance to cefuroxime (cef). whole-genome resequencing revealed only one missense mutation affecting an invariant residue in close proximity to the c-terminal dna-binding domain of rpoc (g1122d). genetic reconstruction experiments demonstrate that this substitution is suffi ...201323070162
twist-joints and double twist-joints in rna structure.analysis of available rna crystal structures has allowed us to identify a new family of rna arrangements that we call double twist-joints, or dtjs. each dtj is composed of a double helix that contains two bulges incorporated into different strands and separated from each other by 2 or 3 bp. at each bulge, the double helix is over-twisted, while the unpaired nucleotides of both bulges form a complex network of stacking and hydrogen-bonding with nucleotides of helical regions. in total, we identif ...201223060425
spectral identification of intermediates generated during the reaction of dioxygen with the wild-type and eq(i-286) mutant of rhodobacter sphaeroides cytochrome c oxidase.cytochrome c oxidase from rhodobacter sphaeroides is frequently used to model the more complex mitochondrial enzyme. the o(2) reduction in both enzymes is generally described by a unidirectional mechanism involving the sequential formation of the ferrous-oxy complex (compound a), the p(r) state, the oxyferryl f form, and the oxidized state. in this study we investigated the reaction of dioxygen with the wild-type reduced r. sphaeroides cytochrome oxidase and the eq(i-286) mutant using the co flo ...201223057757
cloning, expression and bioinformatics analysis of atp sulfurylase from acidithiobacillus ferrooxidans atcc 23270 in escherichia coli.molecular studies of enzymes involved in sulfite oxidation in acidithiobacillus ferrooxidans have not yet been developed, especially in the atp sulfurylase (atps) of these acidophilus tiobacilli that have importance in biomining. this enzyme synthesizes atp and sulfate from adenosine phosphosulfate (aps) and pyrophosphate (ppi), final stage of the sulfite oxidation by these organisms in order to obtain energy. the atps gene (1674 bp) encoding the atps from acidithiobacillus ferrooxidans atcc 232 ...201223055613
alternative ground states enable pathway switching in biological electron transfer.electron transfer is the simplest chemical reaction and constitutes the basis of a large variety of biological processes, such as photosynthesis and cellular respiration. nature has evolved specific proteins and cofactors for these functions. the mechanisms optimizing biological electron transfer have been matter of intense debate, such as the role of the protein milieu between donor and acceptor sites. here we propose a mechanism regulating long-range electron transfer in proteins. specifically ...201223054836
the crystal structures of the α-subunit of the α(2)β (2) tetrameric glycyl-trna synthetase.aminoacyl-trna synthetases (aarss) are ligases (ec.6.1.1.-) that catalyze the acylation of amino acids to their cognate trnas in the process of translating genetic information from mrna to protein. their amino acid and trna specificity are crucial for correctly translating the genetic code. glycine is the smallest amino acid and the glycyl-trna synthetase (glyrs) belongs to class ii aarss. the enzyme is unusual because it can assume different quaternary structures. in eukaryotes, archaebacteria ...201223054484
subunit-specific incorporation efficiency and kinetics in mitochondrial complex i homeostasis.studies employing native page suggest that most ndna-encoded ci subunits form subassemblies before assembling into holo-ci. in addition, in vitro evidence suggests that some subunits can directly exchange in holo-ci. presently, data on the kinetics of these two incorporation modes for individual ci subunits during ci maintenance are sparse. here, we used inducible hek293 cell lines stably expressing acgfp1-tagged ci subunits and quantified the amount of tagged subunit in mitoplasts and holo-ci b ...201223038253
thermal control of microbial development and virulence: molecular mechanisms of microbial temperature sensing.temperature is a critical and ubiquitous environmental signal that governs the development and virulence of diverse microbial species, including viruses, archaea, bacteria, fungi, and parasites. microbial survival is contingent upon initiating appropriate responses to the cellular stress induced by severe environmental temperature change. in the case of microbial pathogens, development and virulence are often coupled to sensing host physiological temperatures. as such, microbes have developed di ...201223033469
mining the sinorhizobium meliloti transportome to develop fret biosensors for sugars, dicarboxylates and cyclic polyols.förster resonance energy transfer (fret) biosensors are powerful tools to detect biologically important ligands in real time. currently fret bisosensors are available for twenty-two compounds distributed in eight classes of chemicals (two pentoses, two hexoses, two disaccharides, four amino acids, one nucleobase, two nucleotides, six ions and three phytoestrogens). to expand the number of available fret biosensors we used the induction profile of the sinorhizobium meliloti transportome to system ...201223028462
convergent transmission of rnai guide-target mismatch information across argonaute internal allosteric network.in rna interference, a guide strand derived from a short dsrna such as a microrna (mirna) is loaded into argonaute, the central protein in the rna induced silencing complex (risc) that silences messenger rnas on a sequence-specific basis. the positions of any mismatched base pairs in an mirna determine which argonaute subtype is used. subsequently, the argonaute-guide complex binds and silences complementary target mrnas; certain argonautes cleave the target. mismatches between guide strand and ...201223028290
high-resolution structures of thermus thermophilus enoyl-acyl carrier protein reductase in the apo form, in complex with nad+ and in complex with nad+ and triclosan.enoyl-acyl carrier protein reductase (enr; the product of the fabi gene) is an important enzyme that is involved in the type ii fatty-acid-synthesis pathway of bacteria, plants, apicomplexan protozoa and mitochondria. harmful pathogens such as mycobacterium tuberculosis and plasmodium falciparum use the type ii fatty-acid-synthesis system, but not mammals or fungi, which contain a type i fatty-acid-synthesis pathway consisting of one or two multifunctional enzymes. for this reason, specific inhi ...201223027736
effect of lecithin on d-galactosamine induced hepatotoxicity through mitochondrial pathway involving bcl-2 and bax.twenty four wistar strain albino rats were used for the investigations. lecithin 50 and 100 mg/kg b wt was administered for 1 week by oral route. liver damage was induced by intra peritoneal administration of 400 mg/kg b wt d-galactosamine on the last day. at the end of the study animals were sacrificed and liver enzyme levels, histopathology, mitochondrial integrity, expression of p53, bax and bcl-2 mrna levels were studied. increases in the liver enzyme levels by d-galn were significantly inhi ...201123024474
a universal method for the identification of bacteria based on general pcr primers.the universal method (um) described here will allow the detection of any bacterial rdna leading to the identification of that bacterium. the method should allow prompt and accurate identification of bacteria. the principle of the method is simple; when a pure pcr product of the 16s gene is obtained, sequenced, and aligned against bacterial dna data base, then the bacterium can be identified. confirmation of identity may follow. in this work, several general 16s primers were designed, mixed and a ...201123024404
unusual c=c bond isomerization of an α,β-unsaturated γ-butyrolactone catalysed by flavoproteins from the old yellow enzyme family.an unexpected, redox-neutral c=c bond isomerization of a γ-butyrolactone bearing an exo-methylene unit to the thermodynamically more favoured endo isomer (k(cat) =0.076 s(-1) ) catalysed by flavoproteins from the old yellow enzyme family was discovered. theoretical calculations and kinetic data support a mechanism through which the isomerization proceeds through fmn-mediated hydride addition onto exo-cβ, followed by hydride abstraction from endo-cβ', which is in line with the well-established c= ...201223024004
functional analysis of three arabidopsis argonautes using slicer-defective mutants.in rna-directed silencing pathways, ternary complexes result from small rna-guided argonaute (ago) associating with target transcripts. target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. here, wild-type and active-site defective forms of several arabidopsis thaliana ago proteins involved in posttranscriptional silencing were used to examine several ago functions, including small rn ...201223023169
basic mechanisms of rna polymerase ii activity and alteration of gene expression in saccharomyces cerevisiae.transcription by rna polymerase ii (pol ii), and all rna polymerases for that matter, may be understood as comprising two cycles. the first cycle relates to the basic mechanism of the transcription process wherein pol ii must select the appropriate nucleoside triphosphate (ntp) substrate complementary to the dna template, catalyze phosphodiester bond formation, and translocate to the next position on the dna template. performing this cycle in an iterative fashion allows the synthesis of rna chai ...201223022618
basic mechanisms of rna polymerase ii activity and alteration of gene expression in saccharomyces cerevisiae.transcription by rna polymerase ii (pol ii), and all rna polymerases for that matter, may be understood as comprising two cycles. the first cycle relates to the basic mechanism of the transcription process wherein pol ii must select the appropriate nucleoside triphosphate (ntp) substrate complementary to the dna template, catalyze phosphodiester bond formation, and translocate to the next position on the dna template. performing this cycle in an iterative fashion allows the synthesis of rna chai ...201223022618
recor complex including recr n-n dimer and reco monomer displays a high affinity for ssdna.recr is an important recombination mediator protein in the recfor pathway. recr together with reco and recf facilitates reca nucleoprotein filament formation and homologous pairing. structural and biochemical studies of thermoanaerobacter tengcongensis recr (tterecr) and its series mutants revealed that tterecr uses the n-n dimer as a basic functional unit to interact with ttereco monomer. two tterecr n-n dimers form a ring-shaped tetramer via an interaction between their c-terminal regions. the ...201223019218
copper starvation-inducible protein for cytochrome oxidase biogenesis in bradyrhizobium japonicum.microarray analysis of bradyrhizobium japonicum grown under copper limitation uncovered five genes named pcuabcde, which are co-transcribed and co-regulated as an operon. the predicted gene products are periplasmic proteins (pcua, pcuc, and pcud), a tonb-dependent outer membrane receptor (pcub), and a cytoplasmic membrane-integral protein (pcue). homologs of pcuc and pcue had been discovered in other bacteria, namely pcu(a)c and ycnj, where they play a role in cytochrome oxidase biogenesis and c ...201223012364
the crystal structure of the rv0301-rv0300 vapbc-3 toxin-antitoxin complex from m. tuberculosis reveals a mg²⁺ ion in the active site and a putative rna-binding site.vapbc pairs account for 45 out of 88 identified toxin-antitoxin (ta) pairs in the mycobacterium tuberculosis (mtb) h37rv genome. a working model suggests that under times of stress, antitoxin molecules are degraded, releasing the toxins to slow the metabolism of the cell, which in the case of vapc toxins is via their rnase activity. otherwise the ta pairs remain bound to their promoters, autoinhibiting transcription. the crystal structure of rv0301-rv0300, an mtb vapbc ta complex determined at 1 ...201223011806
frequency, spectrum, and nonzero fitness costs of resistance to myxopyronin in staphylococcus aureus.the antibiotic myxopyronin (myx) functions by inhibiting bacterial rna polymerase (rnap). the binding site on rnap for myx-the rnap "switch region sw1/sw2 subregion"-is different from the binding site on rnap for the rnap inhibitor currently used in broad-spectrum antibacterial therapy, rifampin (rif). here, we report the frequency, spectrum, and fitness costs of myx resistance in staphylococcus aureus. the resistance rate for myx is 4 × 10(-8) to 7 × 10(-8) per generation, which is equal within ...201223006749
cas5d processes pre-crrna and is a member of a larger family of crispr rna endonucleases.small rnas derived from clustered, regularly interspaced, short palindromic repeat (crispr) loci in bacteria and archaea are involved in an adaptable and heritable gene-silencing pathway. resistance to invasive genetic material is conferred by the incorporation of short dna sequences derived from this material into the genome as crispr spacer elements separated by short repeat sequences. processing of long primary transcripts (pre-crrnas) containing these repeats by a crispr-associated (cas) rna ...201223006625
inactivation of ribosomal protein genes in bacillus subtilis reveals importance of each ribosomal protein for cell proliferation and cell differentiation.among the 57 genes that encode ribosomal proteins in the genome of bacillus subtilis, a gram-positive bacterium, 50 genes were targeted by systematic inactivation. individual deletion mutants of 16 ribosomal proteins (l1, l9, l15, l22, l23, l28, l29, l32, l33.1, l33.2, l34, l35, l36, s6, s20, and s21) were obtained successfully. in conjunction with previous reports, 22 ribosomal proteins have been shown to be nonessential in b. subtilis, at least for cell proliferation. although several mutants ...201223002217
enzyme redesign guided by cancer-derived idh1 mutations.mutations in an enzyme can result in a neomorphic catalytic activity in cancers. we applied cancer-associated mutations from isocitrate dehydrogenases to homologous residues in the active sites of homoisocitrate dehydrogenases to derive enzymes that catalyze the conversion of 2-oxoadipate to (r)-2-hydroxyadipate, a critical step for adipic acid production. thus, we provide a prototypic example of how insights from cancer genome sequencing and functional studies can aid in enzyme redesign.201223001033
mechanisms that impact microrna stability in plants.micrornas (mirnas) are 20-24 nucleotide rnas that regulate a variety of developmental and metabolic processes. the accumulation of mirnas in vivo can be controlled at multiple levels. in addition to mirna biogenesis, mechanisms that lead to rna degradation, such as 3' uridylation and 3' truncation, also affect the steady-state levels of mirnas. on the other hand, 2'-o-methylation in plant mirnas protects their 3' ends from truncation and uridylation. the recent identification of heso1 as the key ...201222995833
the 70s ribosome modulates the atpase activity of escherichia coli ychf.ychf is one of two universally conserved gtpases with unknown cellular function. as a first step toward elucidating ychf's cellular role, we performed a detailed biochemical characterization of the protein from escherichia coli. our data from fluorescence titrations not only confirmed the surprising finding that ychfe.coli binds adenine nucleotides more efficiently than guanine nucleotides, but also provides the first evidence suggesting that ychf assumes two distinct conformational states (atp- ...201222995830
a multicenter blinded analysis indicates no association between chronic fatigue syndrome/myalgic encephalomyelitis and either xenotropic murine leukemia virus-related virus or polytropic murine leukemia virus.the disabling disorder known as chronic fatigue syndrome or myalgic encephalomyelitis (cfs/me) has been linked in two independent studies to infection with xenotropic murine leukemia virus-related virus (xmrv) and polytropic murine leukemia virus (pmlv). although the associations were not confirmed in subsequent studies by other investigators, patients continue to question the consensus of the scientific community in rejecting the validity of the association. here we report blinded analysis of p ...201222991430
crystal structures of complexes of the branched-chain aminotransferase from deinococcus radiodurans with α-ketoisocaproate and l-glutamate suggest the radiation resistance of this enzyme for catalysis.branched-chain aminotransferases (bcat), which utilize pyridoxal 5'-phosphate (plp) as a cofactor, reversibly catalyze the transfer of the α-amino groups of three of the most hydrophobic branched-chain amino acids (bcaa), leucine, isoleucine, and valine, to α-ketoglutarate to form the respective branched-chain α-keto acids and glutamate. the bcat from deinococcus radiodurans (drbcat), an extremophile, was cloned and expressed in escherichia coli for structure and functional studies. the crystal ...201222984263
basic mechanism of transcription by rna polymerase ii.rna polymerase ii-like enzymes carry out transcription of genomes in eukaryota, archaea, and some viruses. they also exhibit fundamental similarity to rna polymerases from bacteria, chloroplasts, and mitochondria. in this review we take an inventory of recent studies illuminating different steps of basic transcription mechanism, likely common for most multi-subunit rna polymerases. through the amalgamation of structural and computational chemistry data we attempt to highlight the most feasible r ...201222982365
basic mechanism of transcription by rna polymerase ii.rna polymerase ii-like enzymes carry out transcription of genomes in eukaryota, archaea, and some viruses. they also exhibit fundamental similarity to rna polymerases from bacteria, chloroplasts, and mitochondria. in this review we take an inventory of recent studies illuminating different steps of basic transcription mechanism, likely common for most multi-subunit rna polymerases. through the amalgamation of structural and computational chemistry data we attempt to highlight the most feasible r ...201222982365
the spt4-spt5 complex: a multi-faceted regulator of transcription elongation.in all domains of life, elongating rna polymerases require the assistance of accessory factors to maintain their processivity and regulate their rate. among these elongation factors, the spt5/nusg factors stand out. members of this protein family appear to be the only transcription accessory proteins that are universally conserved across all domains of life. in archaea and eukaryotes, spt5 associates with a second protein, spt4. in addition to regulating elongation, the eukaryotic spt4-spt5 comp ...201222982195
the spt4-spt5 complex: a multi-faceted regulator of transcription elongation.in all domains of life, elongating rna polymerases require the assistance of accessory factors to maintain their processivity and regulate their rate. among these elongation factors, the spt5/nusg factors stand out. members of this protein family appear to be the only transcription accessory proteins that are universally conserved across all domains of life. in archaea and eukaryotes, spt5 associates with a second protein, spt4. in addition to regulating elongation, the eukaryotic spt4-spt5 comp ...201222982195
benefits of using molecular structure and abundance in phylogenomic analysis. 201222973296
characterization of the genome, proteome, and structure of yersiniophage ϕr1-37.the bacteriophage vb_yecm-ϕr1-37 (ϕr1-37) is a lytic yersiniophage that can propagate naturally in different yersinia species carrying the correct lipopolysaccharide receptor. this large-tailed phage has deoxyuridine (du) instead of thymidine in its dna. in this study, we determined the genomic sequence of phage ϕr1-37, mapped parts of the phage transcriptome, characterized the phage particle proteome, and characterized the virion structure by cryo-electron microscopy and image reconstruction. t ...201222973030
oxygen and hydrogen peroxide in the early evolution of life on earth: in silico comparative analysis of biochemical pathways.in the universe, oxygen is the third most widespread element, while on earth it is the most abundant one. moreover, oxygen is a major constituent of all biopolymers fundamental to living organisms. besides o(2), reactive oxygen species (ros), among them hydrogen peroxide (h(2)o(2)), are also important reactants in the present aerobic metabolism. according to a widely accepted hypothesis, aerobic metabolism and many other reactions/pathways involving o(2) appeared after the evolution of oxygenic ...201222970865
archaeal jab1/mpn/mov34 metalloenzyme (hvjamm1) cleaves ubiquitin-like small archaeal modifier proteins (samps) from protein-conjugates.proteins with jab1/mpn/mov34 metalloenzyme (jamm/mpn+) domains are widespread among all domains of life, yet poorly understood. here we report the purification and characterization of an archaeal jamm/mpn+ domain protein (hvjamm1) from haloferax volcanii that cleaves ubiquitin-like small archaeal modifier proteins (samp1/2) from protein conjugates. hvjamm1 cleaved samp1/2 conjugates generated in h. volcanii as well as isopeptide- and linear-linked samp1-moae in purified form. cleavage of linear ...201222970855
specific metal recognition in nickel trafficking.nickel is an essential metal for a number of bacterial species that have developed systems for acquiring, delivering, and incorporating the metal into target enzymes and controlling the levels of nickel in cells to prevent toxic effects. as with other transition metals, these trafficking systems must be able to distinguish between the desired metal and other transition metal ions with similar physical and chemical properties. because there are few enzymes (targets) that require nickel for activi ...201222970729
the b. subtilis mgte magnesium transporter can functionally compensate trpm7-deficiency in vertebrate b-cells.recent studies have shown that the vertebrate magnesium transporters solute carrier family 41, members 1 and 2 (slc41a1, slc41a2) and magnesium transporter subtype 1 (magt1) can endow vertebrate b-cells lacking the ion-channel kinase transient receptor potential cation channel, subfamily m, member 7 (trpm7) with a capacity to grow and proliferate. slc41a1 and slc41a2 display distant homology to the prokaryotic family of mg(2+) transporters, mgte, first characterized in bacillus subtilis. these s ...201222970223
identification of elongation factor g as the conserved cellular target of argyrin b.argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. here, we identify elongation factor g (ef-g) as the cellular target of argyrin b in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. argyrin b binds a novel allosteric pocket in ef-g, distinct from the known ef-g inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. in euka ...201222970117
naxd is a deacetylase required for lipid a modification and francisella pathogenesis.modification of specific gram-negative bacterial cell envelope components, such as capsule, o-antigen and lipid a, are often essential for the successful establishment of infection. francisella species express lipid a molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. in this study, we show that naxd, a member of the highly conserved ydjc superfamily, is a deacetylase required for an important modification of the oute ...201222966934
common chaperone activity in the g-domain of trgtpase protects l11-l12 interaction on the ribosome.translational gtpases (trgtpases) regulate all phases of protein synthesis. an early event in the interaction of a trgtpase with the ribosome is the contact of the g-domain with the c-terminal domain (ctd) of ribosomal protein l12 (l12-ctd) and subsequently interacts with the n-terminal domain of l11 (l11-ntd). however, the structural and functional relationships between l12-ctd and l11-ntd remain unclear. here, we performed mutagenesis, biochemical and structural studies to identify the interac ...201222965132
decoding properties of trna leave a detectable signal in codon usage bias.the standard genetic code translates 61 codons into 20 amino acids using fewer than 61 transfer rnas (trnas). this is possible because of the trna's ability to 'wobble' at the third base to decode more than one codon. although the anticodon-codon mapping of trna to mrna is a prerequisite for certain codon usage indices and can contribute to the understanding of the evolution of alternative genetic codes, it is usually not determined experimentally because such assays are prohibitively expensive ...201222962450
protein-protein interactions in the β-oxidation part of the phenylacetate utilization pathway: crystal structure of the paaf-paag hydratase-isomerase complex.microbial anaerobic and so-called hybrid pathways for degradation of aromatic compounds contain β-oxidation-like steps. these reactions convert the product of the opening of the aromatic ring to common metabolites. the hybrid phenylacetate degradation pathway is encoded in escherichia coli by the paa operon containing genes for 10 enzymes. previously, we have analyzed protein-protein interactions among the enzymes catalyzing the initial oxidation steps in the paa pathway (grishin, a. m., ajamian ...201222961985
insights into glycogen metabolism in chemolithoautotrophic bacteria from distinctive kinetic and regulatory properties of adp-glucose pyrophosphorylase from nitrosomonas europaea.nitrosomonas europaea is a chemolithoautotroph that obtains energy by oxidizing ammonia in the presence of oxygen and fixes co(2) via the benson-calvin cycle. despite its environmental and evolutionary importance, very little is known about the regulation and metabolism of glycogen, a source of carbon and energy storage. here, we cloned and heterologously expressed the genes coding for two major putative enzymes of the glycogen synthetic pathway in n. europaea, adp-glucose pyrophosphorylase and ...201222961847
evolutionary mechanisms of microbial genomes 2012. 201222957300
insights into phosphate cooperativity and influence of substrate modifications on binding and catalysis of hexameric purine nucleoside phosphorylases.the hexameric purine nucleoside phosphorylase from bacillus subtilis (bspnp233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. in order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexame ...201222957058
cross-complementation study of the flagellar type iii export apparatus membrane protein flhb.the bacterial type iii export apparatus is found in the flagellum and in the needle complex of some pathogenic gram-negative bacteria. in the needle complex its function is to secrete effector proteins for infection into eukaryotic cells. in the bacterial flagellum it exports specific proteins for the building of the flagellum during its assembly. the export apparatus is composed of about five membrane proteins and three soluble proteins. the mechanism of the export apparatus is not fully unders ...201222952860
synthetic metabolic engineering-a novel, simple technology for designing a chimeric metabolic pathway.the integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. however, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. one of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved ...201222950411
structural and catalytic differences between two fadh(2)-dependent monooxygenases: 2,4,5-tcp 4-monooxygenase (tftd) from burkholderia cepacia ac1100 and 2,4,6-tcp 4-monooxygenase (tcpa) from cupriavidus necator jmp134.2,4,5-tcp 4-monooxygenase (tftd) and 2,4,6-tcp 4-monooxygenase (tcpa) have been discovered in the biodegradation of 2,4,5-trichlorophenol (2,4,5-tcp) and 2,4,6-trichlorophenol (2,4,6-tcp). tcpa and tftd belong to the reduced flavin adenine dinucleotide (fadh(2))-dependent monooxygenases and both use 2,4,6-tcp as a substrate; however, the two enzymes produce different end products. tftd catalyzes a typical monooxygenase reaction, while tcpa catalyzes a typical monooxygenase reaction followed by a ...201222949829
structure of the human mterf4-nsun4 protein complex that regulates mitochondrial ribosome biogenesis.proteins crucial for the respiratory chain are translated by the mitochondrial ribosome. mitochondrial ribosome biogenesis is therefore critical for oxidative phosphorylation capacity and disturbances are known to cause human disease. this complex process is evolutionary conserved and involves several rna processing and modification steps required for correct ribosomal rna maturation. we recently showed that a member of the mitochondrial transcription termination factor (mterf) family of protein ...201222949673
crystallization and preliminary x-ray crystallographic analysis of ygjg from escherichia coli.putrescine, one of the polyamines that are found in virtually all living organisms, has been implicated as an important biological material. the protein ygjg is involved in the putrescine-degradation pathway in escherichia coli. the enzyme is a putrescine:2-oxoglutarate aminotransferase that belongs to the class iii aminotransferases. in this study, ygjg from e. coli was overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. diffraction data were collected to 2. ...201222949197
structure of the prolyl-trna synthetase from the eukaryotic pathogen giardia lamblia.the genome of the human intestinal parasite giardia lamblia contains only a single aminoacyl-trna synthetase gene for each amino acid. the giardia prolyl-trna synthetase gene product was originally misidentified as a dual-specificity pro/cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-trna synthetases. the 2.2 å resolution crystal structure of the g. lamblia enzyme p ...201222948920
stable cu(ii) and cu(i) mononuclear intermediates in the assembly of the cua center of thermus thermophilus cytochrome oxidase.cua is a dinuclear mixed-valence center located in subunit 2 of the ba(3)-type cytochrome oxidase from thermus thermophilus. the assembly of this site within the periplasmic membrane is believed to be mediated by the copper chaperones sco and/or pcuac, but the biological mechanisms are still poorly understood, thereby stimulating interest in the mechanisms of cua formation from inorganic ions. the formulation of the cua center as an electron-delocalized cu(1.5)-cu(1.5) system implicates both cu( ...201222946616
a blueprint for a mutationist theory of replicative strand asymmetries formation.in the present review, we summarized current knowledge on replicative strand asymmetries in prokaryotic genomes. a cornerstone for the creation of a theory of their formation has been overviewed. according to our recent works, the probability of nonsense mutation caused by replication-associated mutational pressure is higher for genes from lagging strands than for genes from leading strands of both bacterial and archaeal genomes. lower density of open reading frames in lagging strands can be exp ...201222942675
double-stranded endonuclease activity in bacillus halodurans clustered regularly interspaced short palindromic repeats (crispr)-associated cas2 protein.the crispr (clustered regularly interspaced short palindromic repeats) system is a prokaryotic rna-based adaptive immune system against extrachromosomal genetic elements. cas2 is a universally conserved core crispr-associated protein required for the acquisition of new spacers for crispr adaptation. it was previously characterized as an endoribonuclease with preference for single-stranded (ss)rna. here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the bac ...201222942283
resolving the negative potential side (n-side) water-accessible proton pathway of f-type atp synthase by molecular dynamics simulations.the rotation of f(1)f(o)-atp synthase is powered by the proton motive force across the energy-transducing membrane. the protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane f(o) domain, which is coupled to the atp-producing f(1) domain. the hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved asp/glu at the outer transmembrane helix (tmh). an open question is the proton transfer pat ...201222942277
the role of bacterial enhancer binding proteins as specialized activators of σ54-dependent transcription.bacterial enhancer binding proteins (bebps) are transcriptional activators that assemble as hexameric rings in their active forms and utilize atp hydrolysis to remodel the conformation of rna polymerase containing the alternative sigma factor σ(54). we present a comprehensive and detailed summary of recent advances in our understanding of how these specialized molecular machines function. the review is structured by introducing each of the three domains in turn: the central catalytic domain, the ...201222933558
spectroscopic and kinetic investigation of the fully reduced and mixed valence states of ba3-cytochrome c oxidase from thermus thermophilus: a fourier transform infrared (ftir) and time-resolved step-scan ftir study.the complete understanding of a molecular mechanism of action requires the thermodynamic and kinetic characterization of different states and intermediates. cytochrome c oxidase reduces o(2) to h(2)o, a reaction coupled to proton translocation across the membrane. therefore, it is necessary to undertake a thorough characterization of the reduced form of the enzyme and the determination of the electron transfer processes and pathways between the redox-active centers. in this study fourier transfo ...201222927441
evolution of multiple, mutually orthogonal prolyl-trna synthetase/trna pairs for unnatural amino acid mutagenesis in escherichia coli.the site-specific incorporation of unnatural amino acids (uaas) into proteins in living cells relies on an engineered trna/aminoacyl-trna synthetase (trna/aars) pair, orthogonal to the host cell, to deliver the uaa of choice in response to a unique nonsense or frameshift codon. here we report the generation of mutually orthogonal prolyl-trna/prolyl-trna synthase (prors) pairs derived from an archaebacterial ancestor for use in escherichia coli. by reprogramming the anticodon-binding pocket of py ...201222927411
inhibition of protein synthesis on the ribosome by tildipirosin compared with other veterinary macrolides.tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including mannheimia haemolytica and pasteurella multocida, that cause respiratory tract infections in cattle and swine. here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [ic(50)], 0.23 ± 0.01 μm) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. mutation and methylation at key rrna nucleot ...201222926570
crystal structure of rlmm, the 2'o-ribose methyltransferase for c2498 of escherichia coli 23s rrna.rlmm (ygde) catalyzes the s-adenosyl methionine (adomet)-dependent 2'o methylation of c2498 in 23s ribosomal rna (rrna) of escherichia coli. previous experiments have shown that rlmm is active on 23s rrna from an rlmm knockout strain but not on mature 50s subunits from the same strain. here, we demonstrate rlmm methyltransferase (mtase) activity on in vitro transcribed 23s rrna and its domain v. we have solved crystal structures of e. coli rlmm at 1.9 å resolution and of an rlmm-adomet complex a ...201222923526
biochemical and structural insights into xylan utilization by the thermophilic bacterium caldanaerobius polysaccharolyticus.hemicellulose is the next most abundant plant cell wall component after cellulose. the abundance of hemicellulose such as xylan suggests that their hydrolysis and conversion to biofuels can improve the economics of bioenergy production. in an effort to understand xylan hydrolysis at high temperatures, we sequenced the genome of the thermophilic bacterium caldanaerobius polysaccharolyticus. analysis of the partial genome sequence revealed a gene cluster that contained both hydrolytic enzymes and ...201222918832
native tandem and ion mobility mass spectrometry highlight structural and modular similarities in clustered-regularly-interspaced shot-palindromic-repeats (crispr)-associated protein complexes from escherichia coli and pseudomonas aeruginosa.the crispr/cas (clustered regularly interspaced short palindromic repeats/crispr-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to rna-interference. key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different crispr/cas subtypes. previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demo ...201222918228
analysis of the accessibility of clip bound sites reveals that nucleation of the mirna:mrna pairing occurs preferentially at the 3'-end of the seed match.to find out whether the ago-mirna complex is more sensitive to the accessibility of a particular region inside the seed match, we analyze in detail the accessibility of a wide set of mirna binding sites validated by par-clip and hits-clip experiments. our analysis reveals that nucleotides at the 3'-end of bound seed matches are significantly more accessible than nucleotides at the 5'-end as well as nucleotides at any positions in the unbound seed matches. we show that the accessibility of a sing ...201222915600
yeast trm7 interacts with distinct proteins for critical modifications of the trnaphe anticodon loop.post-transcriptional modification of the trna anticodon loop is critical for translation. yeast trm7 is required for 2'-o-methylation of c(32) and n(34) of trna(phe), trna(trp), and trna(leu(uaa)) to form cm(32) and nm(34), and trm7-δ mutants have severe growth and translation defects, but the reasons for these defects are not known. we show here that overproduction of trna(phe) suppresses the growth defect of trm7-δ mutants, suggesting that the crucial biological role of trm7 is the modificatio ...201222912484
the complete genome sequence of natrinema sp. j7-2, a haloarchaeon capable of growth on synthetic media without amino acid supplements.natrinema sp. j7-2 is an extreme haloarchaeon capable of growing on synthetic media without amino acid supplements. here we report the complete genome sequence of natrinema sp. j7-2 which is composed of a 3,697,626-bp chromosome and a 95,989-bp plasmid pj7-i. this is the first complete genome sequence of a member of the genus natrinema. we demonstrate that natrinema sp. j7-2 can use gluconate, glycerol, or acetate as the sole carbon source and that its genome encodes complete metabolic pathways ...201222911826
interaction of card with rna polymerase mediates mycobacterium tuberculosis viability, rifampin resistance, and pathogenesis.mycobacterium tuberculosis infection continues to cause substantial human suffering. new chemotherapeutic strategies, which require insight into the pathways essential for m. tuberculosis pathogenesis, are imperative. we previously reported that depletion of the card protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. card associates with the rna polymerase (rnap), but it has been unknown which of the diverse functions of card are ...201222904282
distinct states of methionyl-trna synthetase indicate inhibitor binding by conformational selection.to guide development of new drugs targeting methionyl-trna synthetase (metrs) for treatment of human african trypanosomiasis, crystal structure determinations of trypanosoma brucei metrs in complex with its substrate methionine and its intermediate product methionyl-adenylate were followed by those of the enzyme in complex with high-affinity aminoquinolone inhibitors via soaking experiments. drastic changes in conformation of one of the two enzymes in the asymmetric unit allowed these inhibitors ...201222902861
recfor proteins target reca protein to a dna gap with either dna or rna at the 5' terminus: implication for repair of stalled replication forks.the repair of single-stranded gaps in duplex dna by homologous recombination requires the proteins of the recf pathway. the assembly of reca protein onto gapped dna (gdna) that is complexed with the single-stranded dna-binding protein is accelerated by the recf, reco, and recr (recfor) proteins. here, we show the recfor proteins specifically target reca protein to gdna even in the presence of a thousand-fold excess of single-stranded dna (ssdna). the binding constant of recf protein, in the pres ...201222902627
allosteric control of the ribosome by small-molecule antibiotics.protein synthesis is targeted by numerous, chemically distinct antibiotics that bind and inhibit key functional centers of the ribosome. using single-molecule imaging and x-ray crystallography, we show that the aminoglycoside neomycin blocks aminoacyl-transfer rna (aa-trna) selection and translocation as well as ribosome recycling by binding to helix 69 (h69) of 23s ribosomal rna within the large subunit of the escherichia coli ribosome. there, neomycin prevents the remodeling of intersubunit br ...201222902368
c16orf57, a gene mutated in poikiloderma with neutropenia, encodes a putative phosphodiesterase responsible for the u6 snrna 3' end modification.c16orf57 encodes a human protein of unknown function, and mutations in the gene occur in poikiloderma with neutropenia (pn), which is a rare, autosomal recessive disease. interestingly, mutations in c16orf57 were also observed among patients diagnosed with rothmund-thomson syndrome (rts) and dyskeratosis congenita (dc), which are caused by mutations in genes involved in dna repair and telomere maintenance. a genetic screen in saccharomyces cerevisiae revealed that the yeast ortholog of c16orf57, ...201222899009
post-translational oxidative modification and inactivation of mitochondrial complex i in epileptogenesis.mitochondrial oxidative stress and damage have been implicated in the etiology of temporal lobe epilepsy, but whether or not they have a functional impact on mitochondrial processes during epilepsy development (epileptogenesis) is unknown. one consequence of increased steady-state mitochondrial reactive oxygen species levels is protein post-translational modification (ptm). we hypothesize that complex i (ci), a protein complex of the mitochondrial electron transport chain, is a target for oxidan ...201222895709
flexible connection of the n-terminal domain in clpb modulates substrate binding and the aggregate reactivation efficiency.clpb reactivates aggregated proteins in cooperation with dnak/j. the clpb monomer contains two nucleotide-binding domains (d1, d2), a coiled-coil domain, and an n-terminal domain attached to d1 with a 17-residue-long unstructured linker containing a gly-gly motif. the clpb-mediated protein disaggregation is linked to translocation of substrates through the central channel in the hexameric clpb, but the events preceding the translocation are poorly understood. the n-terminal domains form a ring s ...201222890624
molecular dynamics simulations of ago silencing complexes reveal a large repertoire of admissible 'seed-less' targets.to better understand the recognition mechanism of risc and the repertoire of guide-target interactions we introduced g:u wobbles and mismatches at various positions of the microrna (mirna) 'seed' region and performed all-atom molecular dynamics simulations of the resulting ago-mirna:mrna ternary complexes. our simulations reveal that many modifications, including combinations of multiple g:u wobbles and mismatches in the seed region, are admissible and result in only minor structural fluctuation ...201222888400
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