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the putative small terminase from the thermophilic dsdna bacteriophage g20c is a nine-subunit oligomer.the assembly of double-stranded dna bacteriophages is dependent on a small terminase protein that normally plays two important roles. firstly, the small terminase protein specifically recognizes viral dna and recruits the large terminase protein, which makes the initial cut in the dsdna. secondly, once the complex of the small terminase, the large terminase and the dna has docked to the portal protein, and dna translocation into a preformed empty procapsid has begun, the small terminase modulate ...201323908032
structure of dihydrouridine synthase c (dusc) from escherichia coli.dihydrouridine (d) is one of the most widely conserved trna modifications. dihydrouridine synthase (dus) is responsible for introducing d modifications into rna by the reduction of uridine. recently, a unique substrate-recognition mechanism using a small adapter molecule has been proposed for thermus thermophilus dus (tthdusc). to acquire insight regarding its substrate-recognition mechanism, the crystal structure of dusc from escherichia coli (ecodusc) was determined at 2.1 å resolution. ecodus ...201323908023
genome sequence of thermus thermophilus atcc 33923, a thermostable trehalose-producing strain.thermus thermophilus atcc 33923 contains a thermostable enzyme that can efficiently catalyze the conversion of maltose into trehalose. here we report a 2.15-mb assembly of its genome sequence and other useful information, including the coding sequences (cds) responsible for biological processes such as dna replication, dna repair, and rna maturation.201323887916
crystal structure of saccharomyces cerevisiae aro8, a putative α-aminoadipate aminotransferase.α-aminoadipate aminotransferase (aaa-at) catalyzes the amination of 2-oxoadipate to α-aminoadipate in the fourth step of the α-aminoadipate pathway of lysine biosynthesis in fungi. the aromatic aminotransferase aro8 has recently been identified as an aaa-at in saccharomyces cerevisiae. this enzyme displays broad substrate selectivity, utilizing several amino acids and 2-oxo acids as substrates. here we report the 1.91å resolution crystal structure of aro8 and compare it to aaa-at lysn from therm ...201323893908
the ribosome triggers the stringent response by rela via a highly distorted trna.the bacterial stringent response links nutrient starvation with the transcriptional control of genes. this process is initiated by the stringent factor rela, which senses the presence of deacylated trna in the ribosome as a symptom of amino-acid starvation to synthesize the alarmone (p)ppgpp. here we report a cryo-em study of rela bound to ribosomes bearing cognate, deacylated trna in the a-site. the data show that rela on the ribosome stabilizes an unusual distorted form of the trna, with the a ...201323877429
diagnosis of barmah forest virus infection by a nested real-time sybr green rt-pcr assay.barmah forest virus (bfv) is a mosquito borne (+) ssrna alphavirus found only in australia. it causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. we developed a real-time pcr assay to detect bfv in an effort to improve diagnosis early in the course of infection. the limit of detection was 16 genome equivalents with a specificity of 100%. fifty five serum samples from bfv-infected patients were tested by the pcr. 52 of 53 antibody-positive samples were pcr negat ...201323935816
toward repurposing ciclopirox as an antibiotic against drug-resistant acinetobacter baumannii, escherichia coli, and klebsiella pneumoniae.antibiotic-resistant infections caused by gram-negative bacteria are a major healthcare concern. repurposing drugs circumvents the time and money limitations associated with developing new antimicrobial agents needed to combat these antibiotic-resistant infections. here we identified the off-patent antifungal agent, ciclopirox, as a candidate to repurpose for antibiotic use. to test the efficacy of ciclopirox against antibiotic-resistant pathogens, we used a curated collection of acinetobacter b ...201323936064
proline dehydrogenase regulates redox state and respiratory metabolism in trypanosoma cruzi.over the past three decades, l-proline has become recognized as an important metabolite for trypanosomatids. it is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. in addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. in this paper, we demonstrate that l-proline is oxid ...201323894476
simultaneous use of muts and reca for suppression of nonspecific amplification during pcr.thermus thermophilus muts, a thermostable mismatch-recognizing protein, is utilized in pcr to suppress nonspecific amplification by preventing synthesis from mismatched primers. t. thermophilus reca also decreases nonspecific amplification by promoting proper hybridization between the primer and template. we observed that muts and reca function under the same reaction conditions and that muts and reca do not preclude each other. furthermore, there were some dna sequences for which only one of th ...201323970956
combining crystallography and epr: crystal and solution structures of the multidomain cochaperone dnaj.hsp70 chaperones assist in a large variety of protein-folding processes in the cell. crucial for these activities is the regulation of hsp70 by hsp40 cochaperones. dnaj, the bacterial homologue of hsp40, stimulates atp hydrolysis by dnak (hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. the first structure of a complete functional dimeric dnaj was determined and the mobility of its individual domains in solution was investigated. ...201323897477
proteogenomic analysis of a thermophilic bacterial consortium adapted to deconstruct switchgrass.thermophilic bacteria are a potential source of enzymes for the deconstruction of lignocellulosic biomass. however, the complement of proteins used to deconstruct biomass and the specific roles of different microbial groups in thermophilic biomass deconstruction are not well-explored. here we report on the metagenomic and proteogenomic analyses of a compost-derived bacterial consortium adapted to switchgrass at elevated temperature with high levels of glycoside hydrolase activities. near-complet ...201323894306
energy, genes and evolution: introduction to an evolutionary synthesis.life is the harnessing of chemical energy in such a way that the energy-harnessing device makes a copy of itself. no energy, no evolution. the 'modern synthesis' of the past century explained evolution in terms of genes, but this is only part of the story. while the mechanisms of natural selection are correct, and increasingly well understood, they do little to explain the actual trajectories taken by life on earth. from a cosmic perspective-what is the probability of life elsewhere in the unive ...201323754807
evidence of direct complementary interactions between messenger rnas and their cognate proteins.recently, the ability to interact with messenger rna (mrna) has been reported for a number of known rna-binding proteins, but surprisingly also for different proteins without recognizable rna binding domains including several transcription factors and metabolic enzymes. moreover, direct binding to cognate mrnas has been detected for multiple proteins, thus creating a strong impetus to search for functional significance and basic physico-chemical principles behind such interactions. here, we deri ...201323868089
the catalytic domain of topological knot trna methyltransferase (trmh) discriminates between substrate trna and nonsubstrate trna via an induced-fit process.a conserved guanosine at position 18 (g18) in the d-loop of trnas is often modified to 2'-o-methylguanosine (gm). formation of gm18 in eubacterial trna is catalyzed by trna (gm18) methyltransferase (trmh). trmh enzymes can be divided into two types based on their substrate trna specificity. type i trmh, including thermus thermophilus trmh, can modify all trna species, whereas type ii trmh, for example escherichia coli trmh, modifies only a subset of trna species. our previous crystal study showe ...201323867454
subunit δ is the key player for assembly of the h(+)-translocating unit of escherichia coli f(o)f1 atp synthase.the atp synthase (f(o)f1) of escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of atp. this nanomotor is composed of the rotor c10γε and the stator ab2α3β3δ. to study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled f(o)f1 complexes by aff ...201323864656
energy transducing roles of antiporter-like subunits in escherichia coli ndh-1 with main focus on subunit nuon (nd2).the proton-translocating nadh-quinone oxidoreductase (complex i/ndh-1) contains a peripheral and a membrane domain. three antiporter-like subunits in the membrane domain, nuol, nuom, and nuon (nd5, nd4 and nd2, respectively), are structurally similar. we analyzed the role of nuon in escherichia coli ndh-1. the lysine residue at position 395 in nuon (nlys(395)) is conserved in nuol (llys(399)) but is replaced by glutamic acid (mglu(407)) in nuom. our mutation study on nlys(395) suggests that this ...201323864658
the deoxynucleotide triphosphohydrolase samhd1 is a major regulator of dna precursor pools in mammalian cells.sterile alpha motif and hd-domain containing protein 1 (samhd1) is a triphosphohydrolase converting deoxynucleoside triphosphates (dntps) to deoxynucleosides. the enzyme was recently identified as a component of the human innate immune system that restricts hiv-1 infection by removing dntps required for viral dna synthesis. samhd1 has deep evolutionary roots and is ubiquitous in human organs. here we identify a general function of samhd1 in the regulation of dntp pools in cultured human cells. t ...201323858451
the human flavoproteome.vitamin b2 (riboflavin) is an essential dietary compound used for the enzymatic biosynthesis of fmn and fad. the human genome contains 90 genes encoding for flavin-dependent proteins, six for riboflavin uptake and transformation into the active coenzymes fmn and fad as well as two for the reduction to the dihydroflavin form. flavoproteins utilize either fmn (16%) or fad (84%) while five human flavoenzymes have a requirement for both fmn and fad. the majority of flavin-dependent enzymes catalyze ...201323500531
crucial optimization of translational components towards efficient incorporation of unnatural amino acids into proteins in mammalian cells.the ability to site-specifically incorporate unnatural amino acids (uaas) into proteins is a powerful tool in protein engineering. while dozens of uaas have been successfully introduced into proteins expressed by escherichia coli cells, it has been much more challenging to create trna and trna-synthetase pairs that enable uaas incorporation, for use in mammalian systems. by altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in e. coli could be use ...201323874413
translocation and fidelity of escherichia coli rna polymerase.exonuclease (exo) iii was used as a probe of the escherichia coli rna polymerase (rnap) ternary elongation complex (tec) downstream border. in the absence of ntps, rnap appears to stall primarily in a post-translocated state and to return slowly to a pre-translocated state. exo iii mapping, therefore, appears inconsistent with an unrestrained thermal ratchet model for translocation, in which rnap freely and rapidly oscillates between pre- and post-translocated positions. the forward translocatio ...201323863783
individual interactions of the b subunits within the stator of the escherichia coli atp synthase.fof1 atp synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of atp. during catalysis, the peripheral stalk, composed of two b subunits and subunit δ in escherichia coli, counteracts the torque generated by the rotation of the central stalk. here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. ant ...201323846684
crystal structure of the ligand-binding form of nanornase from bacteroides fragilis, a member of the dhh/dhha1 phosphoesterase family of proteins.nanornase (nrn) specifically degrades nucleoside 3',5'-bisphosphate and the very short rna, nanorna, during the final step of mrna degradation. the crystal structure of nrn in complex with a reaction product gmp was determined. the overall structure consists of two domains that are interconnected by a flexible loop and form a cleft. two mn²⁺ ions are coordinated by conserved residues in the dhh motif of the n-terminal domain. gmp binds near the dhha1 motif region in the c-terminal domain. our st ...201323851074
structural analysis of mitochondrial mutations reveals a role for bigenomic protein interactions in human disease.mitochondria are the energy producing organelles of the cell, and mutations within their genome can cause numerous and often severe human diseases. at the heart of every mitochondrion is a set of five large multi-protein machines collectively known as the mitochondrial respiratory chain (mrc). this cellular machinery is central to several processes important for maintaining homeostasis within cells, including the production of atp. the mrc is unique due to the bigenomic origin of its interacting ...201323874847
kinetic and isotopic characterization of l-proline dehydrogenase from mycobacterium tuberculosis.the monofunctional proline dehydrogenase (prodh) from mycobacterium tuberculosis performs the flavin-dependent oxidation of l-proline to δ(1)-pyrroline-5-carboxylate in the proline catabolic pathway. the prodh gene, prub, was cloned into the pyub1062 vector, and the c-terminal his-tagged 37 kda protein was expressed and purified by nickel affinity chromatography. a steady-state kinetic analysis revealed a ping-pong mechanism with an overall kcat of 33 ± 2 s(-1) and km values of 5.7 ± 0.8 mm and ...201323834473
pathway-based screening strategy for multitarget inhibitors of diverse proteins in metabolic pathways.many virtual screening methods have been developed for identifying single-target inhibitors based on the strategy of "one-disease, one-target, one-drug". the hit rates of these methods are often low because they cannot capture the features that play key roles in the biological functions of the target protein. furthermore, single-target inhibitors are often susceptible to drug resistance and are ineffective for complex diseases such as cancers. therefore, a new strategy is required for enriching ...201323861662
blasticidin s inhibits translation by trapping deformed trna on the ribosome.the antibiotic blasticidin s (blas) is a potent inhibitor of protein synthesis in bacteria and eukaryotes. we have determined a 3.4-å crystal structure of blas bound to a 70s⋅trna ribosome complex and performed biochemical and single-molecule fret experiments to determine the mechanism of action of the antibiotic. we find that blas enhances trna binding to the p site of the large ribosomal subunit and slows down spontaneous intersubunit rotation in pretranslocation ribosomes. however, the antibi ...201323824292
crystal structure of the ubiquitin-like small archaeal modifier protein 2 from haloferax volcanii.the discovery of ubiquitin-like small archaeal modifier protein 2 (samp2) that forms covalent polymeric chains in haloferax volcanii has generated tremendous interest in the function and regulation of this protein. at present, it remains unclear whether the hfx. volcanii modifier protein samp1 has such polyubiquitinating-like activity. although samp1 and samp2 use the same conjugation machinery to modify their target proteins, each can impart distinct functional consequences. to better understan ...201323821306
the role of mitochondrial function and cellular bioenergetics in ageing and disease.mitochondria constitute an important topic of biomedical enquiry (one paper in every 154 indexed in pubmed since 1998 is retrieved by the keyword 'mitochondria') because of widespread recognition of their importance in cell physiology and pathology. mitochondrial dysfunction is widely implicated in ageing and in the diseases of ageing, through dysfunction in adenosine triphosphate (atp) synthesis, ca(2+) homeostasis, central metabolic pathways or radical production. nonetheless, the mechanisms a ...201323786614
biology of extreme radiation resistance: the way of deinococcus radiodurans.the bacterium deinococcus radiodurans is a champion of extreme radiation resistance that is accounted for by a highly efficient protection against proteome, but not genome, damage. a well-protected functional proteome ensures cell recovery from extensive radiation damage to other cellular constituents by molecular repair and turnover processes, including an efficient repair of disintegrated dna. therefore, cell death correlates with radiation-induced protein damage, rather than dna damage, in bo ...201323818498
thermostable group ii intron reverse transcriptase fusion proteins and their use in cdna synthesis and next-generation rna sequencing.mobile group ii introns encode reverse transcriptases (rts) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron rna with high processivity and fidelity. although the latter properties are potentially useful for applications in cdna synthesis and next-generation rna sequencing (rna-seq), group ii intron rts have been difficult to purify free of the intron rna, and their utility as research tools has not been inv ...201323697550
affinity purification of t7 rna transcripts with homogeneous ends using aribo and crispr tags.affinity purification of rna using the aribo tag technology currently provides an ideal approach to quickly prepare rna with 3' homogeneity. here, we explored strategies to also ensure 5' homogeneity of affinity-purified rnas. first, we systematically investigated the effect of starting nucleotides on the 5' heterogeneity of a small sli rna substrate from the neurospora vs ribozyme purified from an sli-aribo precursor. a series of 32 sli rna sequences with variations in the +1 to +3 region was p ...201323657939
unusual base pairing during the decoding of a stop codon by the ribosome.during normal translation, the binding of a release factor to one of the three stop codons (uga, uaa or uag) results in the termination of protein synthesis. however, modification of the initial uridine to a pseudouridine (ψ) allows efficient recognition and read-through of these stop codons by a transfer rna (trna), although it requires the formation of two normally forbidden purine-purine base pairs. here we determined the crystal structure at 3.1 å resolution of the 30s ribosomal subunit in c ...201323812587
crystal structures of ef-g-ribosome complexes trapped in intermediate states of translocation.translocation of messenger and transfer rna (mrna and trna) through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. the crystal structures of three thermus ribosome-trna-mrna-ef-g complexes trapped with β,γ-imidoguanosine 5'-triphosphate (gdpnp) or fusidic acid reveal conformational changes occurring during intermediate states of translocation, including large-scale rotation of the 30s subunit head and body. in all complexes, the trna acceptor ends occ ...201323812722
elongation factor g bound to the ribosome in an intermediate state of translocation.a key step of translation by the ribosome is translocation, which involves the movement of messenger rna (mrna) and transfer rna (trna) with respect to the ribosome. this allows a new round of protein chain elongation by placing the next mrna codon in the a site of the 30s subunit. translocation proceeds through an intermediate state in which the acceptor ends of the trnas have moved with respect to the 50s subunit but not the 30s subunit, to form hybrid states. the guanosine triphosphatase (gtp ...201323812720
the role of aromatic-aromatic interactions in strand-strand stabilization of β-sheets.aromatic-aromatic interactions have long been believed to play key roles in protein structure, folding, and binding functions. however, we still lack full understanding of the contributions of aromatic-aromatic interactions to protein stability and the timing of their formation during folding. here, using an aromatic ladder in the β-barrel protein, cellular retinoic acid-binding protein 1 (crabp1), as a case study, we find that aromatic π stacking plays a greater role in the phe65-phe71 cross-st ...201323810905
crispr interference: a structural perspective.crispr (cluster of regularly interspaced palindromic repeats) is a prokaryotic adaptive defence system, providing immunity against mobile genetic elements such as viruses. genomically encoded crrna (crispr rna) is used by cas (crispr-associated) proteins to target and subsequently degrade nucleic acids of invading entities in a sequence-dependent manner. the process is known as 'interference'. in the present review we cover recent progress on the structural biology of the crispr/cas system, focu ...201323805973
molecular crowding enhanced atpase activity of the rna helicase eif4a correlates with compaction of its quaternary structure and association with eif4g.enzymatic reactions occur in a crowded and confined environment in vivo, containing proteins, rna and dna. previous reports have shown that interactions between macromolecules, and reactions rates differ significantly between crowded environments and dilute buffers. however, the direct effect of crowding on the level of high-resolution structures of macromolecules has not been extensively analyzed and is not well understood. here we analyze the effect of macromolecular crowding on structure and ...201323767688
an 'open' structure of the recor complex supports ssdna binding within the core of the complex.efficient dna repair is critical for cell survival and the maintenance of genome integrity. the homologous recombination pathway is responsible for the repair of dna double-strand breaks within cells. initiation of this pathway in bacteria can be carried out by either the recbcd or the recfor proteins. an important regulatory player within the recfor pathway is the recor complex that facilitates reca loading onto dna. here we report new data regarding the assembly of deinococcus radiodurans reco ...201323814185
eukaryote-specific insertion elements control human argonaute slicer activity.we have solved the crystal structure of human argonaute1 (hago1) bound to endogenous 5'-phosphorylated guide rnas. to identify changes that evolutionarily rendered hago1 inactive, we compared our structure with guide-rna-containing and cleavage-active hago2. aside from mutation of a catalytic tetrad residue, proline residues at positions 670 and 675 in hago1 introduce a kink in the cs7 loop, forming a convex surface within the hago1 nucleic-acid-binding channel near the inactive catalytic site. ...201323809764
heritable change caused by transient transcription errors.transmission of cellular identity relies on the faithful transfer of information from the mother to the daughter cell. this process includes accurate replication of the dna, but also the correct propagation of regulatory programs responsible for cellular identity. errors in dna replication (mutations) and protein conformation (prions) can trigger stable phenotypic changes and cause human disease, yet the ability of transient transcriptional errors to produce heritable phenotypic change ('epimuta ...201323825966
structural insight into negative dna supercoiling by dna gyrase, a bacterial type 2a dna topoisomerase.type 2a dna topoisomerases (topo2a) remodel dna topology during replication, transcription and chromosome segregation. these multisubunit enzymes catalyze the transport of a double-stranded dna through a transient break formed in another duplex. the bacterial dna gyrase, a target for broad-spectrum antibiotics, is the sole topo2a enzyme able to introduce negative supercoils. we reveal here for the first time the architecture of the full-length thermus thermophilus dna gyrase alone and in a cleav ...201323804759
structural basis for s-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor b1.eukaryotic transcription factor b (tfb) proteins are homologous to ksga/dim1 ribosomal rna (rrna) methyltransferases. the mammalian tfb1, mitochondrial (tfb1m) factor is an essential protein necessary for mitochondrial gene expression. tfb1m mediates an rrna modification in the small ribosomal subunit and thus plays a role analogous to ksga/dim1 proteins. this modification has been linked to mitochondrial dysfunctions leading to maternally inherited deafness, aminoglycoside sensitivity and diabe ...201323804760
the trna recognition mechanism of the minimalist spout methyltransferase, trml.unlike other transfer rnas (trna)-modifying enzymes from the spout methyltransferase superfamily, the trna (um34/cm34) methyltransferase trml lacks the usual extension domain for trna binding and consists only of a spout domain. both the catalytic and trna recognition mechanisms of this enzyme remain elusive. by using trnas purified from an escherichia coli strain with the trml gene deleted, we found that trml can independently catalyze the methyl transfer from s-adenosyl-l-methionine to and iso ...201323804755
co-expression of rna-protein complexes in escherichia coli and applications to rna biology.rna has emerged as a major player in many cellular processes. understanding these processes at the molecular level requires homogeneous rna samples for structural, biochemical and pharmacological studies. we previously devised a generic approach that allows efficient in vivo expression of recombinant rna in escherichia coli. in this work, we have extended this method to rna/protein co-expression. we have engineered several plasmids that allow overexpression of rna-protein complexes in e. coli. w ...201323804766
development of a d-xylose fermenting and inhibitor tolerant industrial saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering.the production of bioethanol from lignocellulose hydrolysates requires a robust, d-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. the purpose of the present work was to develop such a strain from a prime industrial yeast strain, ethanol red, used for bioethanol production.201323800147
kinetic proofreading at single molecular level: aminoacylation of trna(ile) and the role of water as an editor.proofreading/editing in protein synthesis is essential for accurate translation of information from the genetic code. in this article we present a theoretical investigation of efficiency of a kinetic proofreading mechanism that employs hydrolysis of the wrong substrate as the discriminatory step in enzyme catalytic reactions. we consider aminoacylation of trna(ile) which is a crucial step in protein synthesis and for which experimental results are now available. we present an augmented kinetic s ...201323840412
coordination between apoplastic and symplastic detoxification confers plant aluminum resistance.whether aluminum toxicity is an apoplastic or symplastic phenomenon is still a matter of debate. here, we found that three auxin overproducing mutants, yucca, the recessive mutant superroot2, and superroot1 had increased aluminum sensitivity, while a transfer dna insertion mutant, xyloglucan endotransglucosylase/hydrolases15 (xth15), showed enhanced aluminum resistance, accompanied by low endogenous indole-3-acetic acid levels, implying that auxin may be involved in plant responses to aluminum s ...201323776189
dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates.the efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. we used dna-barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both zymomonas mobilis (44 genes) and saccharomyces cerevisiae (99 genes). overexpression of a z. mobilis tolerance gene of unknown functi ...201323774757
expanded use of sense codons is regulated by modified cytidines in trna.codon use among the three domains of life is not confined to the universal genetic code. with only 22 trna genes in mammalian mitochondria, exceptions from the universal code are necessary for proper translation. a particularly interesting deviation is the decoding of the isoleucine aua codon as methionine by the one mitochondrial-encoded trna(met). this trna decodes aua and aug in both the a- and p-sites of the metazoan mitochondrial ribosome. enrichment of posttranscriptional modifications is ...201323781103
multiple pathways promote dynamical coupling between catalytic domains in escherichia coli prolyl-trna synthetase.aminoacyl-trna synthetases are multidomain enzymes that catalyze covalent attachment of amino acids to their cognate trna. cross-talk between functional domains is a prerequisite for this process. in this study, we investigate the molecular mechanism of site-to-site communication in escherichia coli prolyl-trna synthetase (ec prors). earlier studies have demonstrated that evolutionarily conserved and/or co-evolved residues that are engaged in correlated motion are critical for the propagation of ...201323731272
experimental evidence for the thermophilicity of ancestral life.theoretical studies have focused on the environmental temperature of the universal common ancestor of life with conflicting conclusions. here we provide experimental support for the existence of a thermophilic universal common ancestor. we present the thermal stabilities and catalytic efficiencies of nucleoside diphosphate kinases (ndk), designed using the information contained in predictive phylogenetic trees, that seem to represent the last common ancestors of archaea and of bacteria. these en ...201323776221
antituberculosis thiophenes define a requirement for pks13 in mycolic acid biosynthesis.we report a new class of thiophene (tp) compounds that kill mycobacterium tuberculosis by the previously uncharacterized mechanism of pks13 inhibition. an f79s mutation near the catalytic ser55 site in pks13 conferred tp resistance in m. tuberculosis. overexpression of wild-type pks13 resulted in tp resistance, and overexpression of the pks13(f79s) mutant conferred high resistance. in vitro, tp inhibited fatty acyl-amp loading onto pks13. tp inhibited mycolic acid biosynthesis in wild-type m. tu ...201323770708
secondary structure and domain architecture of the 23s and 5s rrnas.we present a de novo re-determination of the secondary (2°) structure and domain architecture of the 23s and 5s rrnas, using 3d structures, determined by x-ray diffraction, as input. in the traditional 2° structure, the center of the 23s rrna is an extended single strand, which in 3d is seen to be compact and double helical. accurately assigning nucleotides to helices compels a revision of the 23s rrna 2° structure. unlike the traditional 2° structure, the revised 2° structure of the 23s rrna sh ...201323771137
crystal structure of the 70s ribosome bound with the q253p mutant form of release factor rf2.bacterial translation termination is mediated by release factors rf1 and rf2, which recognize stop codons and catalyze hydrolysis of the peptidyl-trna ester bond. the catalytic mechanism has been debated. we proposed that the backbone amide nh group, rather than the side chain, of the glutamine of the universally conserved ggq motif participates in catalysis by h-bonding to the tetrahedral transition-state intermediate and by product stabilization. this was supported by complete loss of rf1 cata ...201323769667
extremophilic shmts: from structure to biotechnology.recent advances in molecular and structural biology have improved the availability of virtually any biocatalyst in large quantity and have also provided an insight into the detailed structure-function relationships of many of them. these results allowed the rational exploitation of biocatalysts for use in organic synthesis. in this context, extremophilic enzymes are extensively studied for their potential interest for many biotechnological and industrial applications, as they offer increased rat ...201323841096
in vitro reconstitution of an escherichia coli rna-guided immune system reveals unidirectional, atp-dependent degradation of dna target.many prokaryotes utilize small rna transcribed from clustered, regularly interspaced, short palindromic repeats (crisprs) to protect themselves from foreign genetic elements, such as phage and plasmids. in escherichia coli, this small rna is packaged into a surveillance complex (cascade) that uses the rna sequence to direct binding to invasive dna. once bound, cascade recruits the cas3 nuclease-helicase, which then proceeds to progressively degrade the invading dna. here, using individually puri ...201323760266
structure and function of allophanate hydrolase.allophanate hydrolase converts allophanate to ammonium and carbon dioxide. it is conserved in many organisms and is essential for their utilization of urea as a nitrogen source. it also has important functions in a newly discovered eukaryotic pyrimidine nucleic acid precursor degradation pathway, the yeast-hypha transition that several pathogens utilize to escape the host defense, and an s-triazine herbicide degradation pathway recently emerged in many soil bacteria. we have determined the cryst ...201323754281
structure and function of the t-loop structural motif in noncoding rnas.the t-loop is a frequently occurring five-nucleotide motif found in the structure of noncoding rnas where it is commonly assumed to play an important role in stabilizing the tertiary rna structure by facilitating long-range interactions between different regions of the molecule. t-loops were first identified in trna(phe) and a formal consensus sequence for this motif was formulated and later revised based on analyses of the crystal structures of prokaryotic ribosomal rnas and rnase p and the cor ...201323754657
structural determinants of oligomerization of δ(1)-pyrroline-5-carboxylate dehydrogenase: identification of a hexamerization hot spot.the aldehyde dehydrogenase (aldh) superfamily member δ(1)-pyrroline-5-carboxylate dehydrogenase (p5cdh) catalyzes the nad(+)-dependent oxidation of glutamate semialdehyde to glutamate, which is the final step of proline catabolism. defects in p5cdh activity lead to the metabolic disorder type ii hyperprolinemia, p5cdh is essential for virulence of the fungal pathogen cryptococcus neoformans, and bacterial p5cdhs have been targeted for vaccine development. although the enzyme oligomeric state is ...201323747974
the making of a slicer: activation of human argonaute-1.argonautes are the central protein component in small rna silencing pathways. of the four human argonautes (hago1-hago4) only hago2 is an active slicer. we determined the structure of hago1 bound to endogenous copurified rnas to 1.75 å resolution and hago1 loaded with let-7 microrna to 2.1 å. both structures are strikingly similar to the structures of hago2. a conserved catalytic tetrad within the piwi domain of hago2 is required for its slicing activity. completion of the tetrad, combined with ...201323746446
thermal and spectroscopic characterization of a proton pumping rhodopsin from an extreme thermophile.so far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. in this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium thermus thermophilus, which lives in a hot spring at around 75 °c. the gene for the retinylidene protein, named thermophilic rhodopsin (tr), was chemically synthesized with codon optimization. the codon optimized tr protein was functionally expressed in the cell membranes of escherichia ...201323740255
cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from plasmodium falciparum.isoprenoids are the most diverse and abundant group of natural products. in plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (fpps), turning this enzyme into a key branch point of the isoprenoid synthesis. changes in fpps activity could alter the flux of isoprenoid compounds downstream of fpps and, hence, play a central role in the regulation of a number of essential fu ...201323734739
correlation spectroscopy and molecular dynamics simulations to study the structural features of proteins.in this work, we used a combination of fluorescence correlation spectroscopy (fcs) and molecular dynamics (md) simulation methodologies to acquire structural information on ph-induced unfolding of the maltotriose-binding protein from thermus thermophilus (male2). fcs has emerged as a powerful technique for characterizing the dynamics of molecules and it is, in fact, used to study molecular diffusion on timescale of microsecond and longer. our results showed that keeping temperature constant, the ...201323750215
defining the escherichia coli seca dimer interface residues through in vivo site-specific photo-cross-linking.the motor protein seca is a core component of the bacterial general secretory (sec) pathway and is essential for cell viability. despite evidence showing that seca exists in a dynamic monomer-dimer equilibrium favoring the dimeric form in solution and in the cytoplasm, there is considerable debate as to the quaternary structural organization of the seca dimer. here, a site-directed photo-cross-linking technique was utilized to identify residues on the escherichia coli seca (ecseca) dimer interfa ...201323585536
escherichia coli rimm and yjeq null strains accumulate immature 30s subunits of similar structure and protein complement.assembly of the escherichia coli 30s ribosomal subunits proceeds through multiple parallel pathways. the protein factors rimm, yjeq, rbfa, and era work in conjunction to assist at the late stages of the maturation process of the small subunit. however, it is unclear how the functional interplay between these factors occurs in the context of multiple parallel pathways. to understand how these factors work together, we have characterized the immature 30s subunits that accumulate in δrimm cells and ...201323611982
a new genomic tool, ultra-frequently cleaving taqii/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse dna libraries.genomics and metagenomics are currently leading research areas, with dna sequences accumulating at an exponential rate. although enormous advances in dna sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. a number of dna fragmentation methods, such as hydrodynamic sharing, sonication or dnase i fragmentation, have various drawbacks, including dna damage, poor fragmentation control, irrepr ...201323724933
alternative excision repair pathways.alternative excision repair (aer) is a category of excision repair initiated by a single nick, made by an endonuclease, near the site of dna damage, and followed by excision of the damaged dna, repair synthesis, and ligation. the ultraviolet (uv) damage endonuclease in fungi and bacteria introduces a nick immediately 5' to various types of uv damage and initiates its excision repair that is independent of nucleotide excision repair (ner). endo iv-type apurinic/apyrimidinic (ap) endonucleases fro ...201323645854
improved transferase/hydrolase ratio through rational design of a family 1 β-glucosidase from thermotoga neapolitana.alkyl glycosides are attractive surfactants because of their high surface activity and good biodegradability and can be produced from renewable resources. through enzymatic catalysis, one can obtain well-defined alkyl glycosides, something that is very difficult to do using conventional chemistry. however, there is a need for better enzymes to get a commercially feasible process. a thermostable β-glucosidase from the well-studied glycoside hydrolase family 1 from thermotoga neapolitana, tnbgl1a, ...201323524680
r3d align web server for global nucleotide to nucleotide alignments of rna 3d structures.the r3d align web server provides online access to 'rna 3d align' (r3d align), a method for producing accurate nucleotide-level structural alignments of rna 3d structures. the web server provides a streamlined and intuitive interface, input data validation and output that is more extensive and easier to read and interpret than related servers. the r3d align web server offers a unique gallery of featured alignments, providing immediate access to pre-computed alignments of large rna 3d structures, ...201323716643
common evolutionary origin for the rotor domain of rotary atpases and flagellar protein export apparatus.the v1- and f1- rotary atpases contain a rotor that rotates against a catalytic a3b3 or α3β3 stator. the rotor f(1-γ) or v1-df is composed of both anti-parallel coiled coil and globular-loop parts. the bacterial flagellar type iii export apparatus contains a v1/f1-like atpase ring structure composed of flii6 homo-hexamer and flij which adopts an anti-parallel coiled coil structure without the globular-loop part. here we report that flij of salmonella enterica serovar typhimurium shows a rotor li ...201323724081
cardioprotection by s-nitrosation of a cysteine switch on mitochondrial complex i.oxidative damage from elevated production of reactive oxygen species (ros) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. the mechanism by which the increase in ros occurs is not known, and it is unclear how this increase can be prevented. a wide variety of nitric oxide donors and s-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear. here we used a mitochondria-selective s-nitrosating agent, mitosno, to ...201323708290
structure of an a-form rna duplex obtained by degradation of 6s rna in a crystallization droplet.in the course of a crystallographic study of a 132 nt variant of aquifex aeolicus 6s rna, a crystal structure of an a-form rna duplex containing 12 base pairs was solved at a resolution of 2.6 å. in fact, the rna duplex is part of the 6s rna and was obtained by accidental but precise degradation of the 6s rna in a crystallization droplet. 6s rna degradation was confirmed by microscopic observation of crystals and gel electrophoresis of crystallization droplets. the rna oligomers obtained form re ...201323722840
cryo-em structures of the late-stage assembly intermediates of the bacterial 50s ribosomal subunit.ribosome assembly is a process fundamental for all cellular activities. the efficiency and accuracy of the subunit assembly are tightly regulated and closely monitored. in the present work, we characterized, both compositionally and structurally, a set of in vivo 50s subunit precursors (45s), isolated from a mutant bacterial strain. our qualitative mass spectrometry data indicate that l28, l16, l33, l36 and l35 are dramatically underrepresented in the 45s particles. this protein spectrum shows i ...201323700310
investigation of nadh binding, hydride transfer, and nad(+) dissociation during nadh oxidation by mitochondrial complex i using modified nicotinamide nucleotides.nadh:ubiquinone oxidoreductase (complex i) is a complicated respiratory enzyme that conserves the energy from nadh oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. during catalysis, nadh oxidation by a flavin mononucleotide is followed by electron transfer to a chain of iron-sulfur clusters. alternatively, the flavin may be reoxidized by hydrophilic electron acceptors, by artificial electron acceptors in kinetic studies, or by oxygen a ...201323683271
statistical evaluation of the rodin-ohno hypothesis: sense/antisense coding of ancestral class i and ii aminoacyl-trna synthetases.we tested the idea that ancestral class i and ii aminoacyl-trna synthetases arose on opposite strands of the same gene. we assembled excerpted 94-residue urgenes for class i tryptophanyl-trna synthetase (trprs) and class ii histidyl-trna synthetase (hisrs) from a diverse group of species, by identifying and catenating three blocks coding for secondary structures that position the most highly conserved, active-site residues. the codon middle-base pairing frequency was 0.35 ± 0.0002 in all-by-all ...201323576570
role of loops connecting secondary structure elements in the stabilization of proteins isolated from thermophilic organisms.it has been recently discovered that the connection of secondary structure elements (ββ-unit, βα- and αβ-units) in proteins follows quite stringent principles regarding the chirality and the orientation of the structural units (koga et al., nature 2012;491:222-227). by exploiting these rules, a number of protein scaffolds endowed with a remarkable thermal stability have been designed (koga et al., nature 2012;491:222-227). by using structural databases of proteins isolated from either mesophilic ...201323661276
structure of a dimeric crenarchaeal cas6 enzyme with an atypical active site for crispr rna processing.the competition between viruses and hosts is played out in all branches of life. many prokaryotes have an adaptive immune system termed 'crispr' (clustered regularly interspaced short palindromic repeats) which is based on the capture of short pieces of viral dna. the captured dna is integrated into the genomic dna of the organism flanked by direct repeats, transcribed and processed to generate crrna (crispr rna) that is loaded into a variety of effector complexes. these complexes carry out sequ ...201323527601
bacteriophage p23-77 capsid protein structures reveal the archetype of an ancient branch from a major virus lineage.it has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. however, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. bacteriophage p23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. we have solved multiple crystal structures of the major capsid proteins vp16 and vp ...201323623731
tetramerization reinforces the dimer interface of mnsod.two yeast manganese superoxide dismutases (mnsod), one from saccharomyces cerevisiae mitochondria (scmnsod) and the other from candida albicans cytosol (camnsodc), have most biochemical and biophysical properties in common, yet scmnsod is a tetramer and camnsodc is a dimer or "loose tetramer" in solution. although camnsodc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of camnsodc in vivo depends upon the formation of the tetram ...201323667478
tertiary structure of bacterial selenocysteine trna.selenocysteine (sec) is translationally incorporated into proteins in response to the uga codon. the trna specific to sec (trna(sec)) is first ligated with serine by seryl-trna synthetase (serrs). in the present study, we determined the 3.1 å crystal structure of the trna(sec) from the bacterium aquifex aeolicus, in complex with the heterologous serrs from the archaeon methanopyrus kandleri. the bacterial trna(sec) assumes the l-shaped structure, from which the long extra arm protrudes. although ...201323649835
identification of the nature of reading frame transitions observed in prokaryotic genomes.our goal was to identify evolutionary conserved frame transitions in protein coding regions and to uncover an underlying functional role of these structural aberrations. we used the ab initio frameshift prediction program, genetack, to detect reading frame transitions in 206 991 genes (fs-genes) from 1106 complete prokaryotic genomes. we grouped 102 731 fs-genes into 19 430 clusters based on sequence similarity between protein products (fs-proteins) as well as conservation of predicted position ...201323649834
secretome analysis defines the major role of secdf in staphylococcus aureus virulence.the sec pathway plays a prominent role in protein export and membrane insertion, including the secretion of major bacterial virulence determinants. the accessory sec constituent secdf has been proposed to contribute to protein export. deletion of staphylococcus aureus secdf has previously been shown to reduce resistance, to alter cell separation, and to change the expression of certain virulence factors. to analyse the impact of the secdf deletion in s. aureus on protein secretion, a quantitativ ...201323658837
camphor pathway redux: functional recombinant expression of 2,5- and 3,6-diketocamphane monooxygenases of pseudomonas putida atcc 17453 with their cognate flavin reductase catalyzing baeyer-villiger reactions.whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-dkcmo and 3,6-dkcmo, respectively) in the initial catabolic steps of (+) and (-) isomeric forms of camphor (cam) metabolism in pseudomonas putida atcc 17453 are relatively well characterized, the actual identity of the flavin reductase (fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. in this study, a 37- ...201323524667
cloning, expression, purification and preliminary x-ray diffraction studies of a mycobacterial protein implicated in bacterial survival in macrophages.mycobacterium species have developed numerous strategies to avoid the antimycobacterial actions of macrophages, enabling them to survive within the generally inhospitable environment of the cell. the recently identified msmeg_5817 protein from m. smegmatis is highly conserved in mycobacterium spp. and is required for bacterial survival in macrophages. here, the cloning, expression, purification and crystallization of msmeg_5817 is reported. crystals of msmeg_5817 were grown in 1.42 m li2so4, 0.1 ...201323695579
identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis.the molybdenum cofactor (moco) is a redox cofactor found in all kingdoms of life, and its biosynthesis is essential for survival of many organisms, including humans. the first step of moco biosynthesis is a unique transformation of guanosine 5'-triphosphate (gtp) into cyclic pyranopterin monophosphate (cpmp). in bacteria, moaa and moac catalyze this transformation, although the specific functions of these enzymes were not fully understood. here, we report the first isolation and structural chara ...201323627491
reorganization of an intersubunit bridge induced by disparate 16s ribosomal ambiguity mutations mimics an ef-tu-bound state.after four decades of research aimed at understanding trna selection on the ribosome, the mechanism by which ribosomal ambiguity (ram) mutations promote miscoding remains unclear. here, we present two x-ray crystal structures of the thermus thermophilus 70s ribosome containing 16s rrna ram mutations, g347u and g299a. each of these mutations causes miscoding in vivo and stimulates elongation factor thermo unstable (ef-tu)-dependent gtp hydrolysis in vitro. mutation g299a is located near the inter ...201323630274
structures of mycobacterium tuberculosis fadd10 protein reveal a new type of adenylate-forming enzyme.mycobacterium tuberculosis has a group of 34 fadd proteins that belong to the adenylate-forming superfamily. they are classified as either fatty acyl-amp ligases (faals) or fatty acyl-coa ligases based on sequence analysis. fadd10, involved in the synthesis of a virulence-related lipopeptide, was mis-annotated as a fatty acyl-coa ligase; however, it is in fact a faal that transfers fatty acids to an acyl carrier protein (rv0100). in this study, we have determined the structures of fadd10 in both ...201323625916
two crispr-cas systems in methanosarcina mazei strain gö1 display common processing features despite belonging to different types i and iii.the clustered regularly interspaced short palindromic repeats (crispr) system represents a highly adaptive and heritable defense system against foreign nucleic acids in bacteria and archaea. we analyzed the two crispr-cas systems in methanosarcina mazei strain gö1. although belonging to different subtypes (i-b and iii-b), the leaders and repeats of both loci are nearly identical. also, despite many point mutations in each array, a common hairpin motif was identified in the repeats by a bioinform ...201323619576
how to achieve high-level expression of microbial enzymes: strategies and perspectives.microbial enzymes have been used in a large number of fields, such as chemical, agricultural and biopharmaceutical industries. the enzyme production rate and yield are the main factors to consider when choosing the appropriate expression system for the production of recombinant proteins. recombinant enzymes have been expressed in bacteria (e.g., escherichia coli, bacillus and lactic acid bacteria), filamentous fungi (e.g., aspergillus) and yeasts (e.g., pichia pastoris). the favorable and very a ...201323686280
differential regulation by ppgpp versus pppgpp in escherichia coli.both ppgpp and pppgpp are thought to function collectively as second messengers for many complex cellular responses to nutritional stress throughout biology. there are few indications that their regulatory effects might be different; however, this question has been largely unexplored for lack of an ability to experimentally manipulate the relative abundance of ppgpp and pppgpp. here, we achieve preferential accumulation of either ppgpp or pppgpp with escherichia coli strains through induction of ...201323620295
recognition of two distinct elements in the rna substrate by the rna-binding domain of the t. thermophilus dead box helicase hera.dead box helicases catalyze the atp-dependent destabilization of rna duplexes. whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the rna substrate. the thermus thermophilus dead-box helicase hera (for "heat-resistant rna-binding atpase") contains a c-terminal rna-binding domain (rbd). we have analyzed rna binding to the hera rbd by a combination of mutational analyses, nuclear magnetic resonance and x-r ...201323625962
the mechanism of e. coli rna polymerase regulation by ppgpp is suggested by the structure of their complex.guanosine tetraphosphate (ppgpp) is an alarmone that enables bacteria to adapt to their environment. it has been known for years that ppgpp acts directly on rna polymerase (rnap) to alter the rate of transcription, but its exact target site is still under debate. here we report a crystal structure of escherichia coli rnap holoenzyme in complex with ppgpp at 4.5 å resolution. the structure reveals that ppgpp binds at an interface between the shelf and core modules on the outer surface of rnap, aw ...201323623685
stable expression plasmids for streptomyces based on a toxin-antitoxin system.bacteria included in the genus streptomyces exhibit several attractive characteristics that make them adequate hosts for the heterologous expression of proteins. one of them is that some of its species have a high secretion capacity and hence the protein of interest could be released to the culture supernatant, facilitating downstream processing. to date, all the expression vectors described for these bacteria contain antibiotic resistance genes as selection markers. however, the use of antibiot ...201323617558
the magic spot: a ppgpp binding site on e. coli rna polymerase responsible for regulation of transcription initiation.the global regulatory nucleotide ppgpp ("magic spot") regulates transcription from a large subset of escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with rna polymerase (rnap) without binding to dna. however, ppgpp's target site on rnap, and therefore its mechanism of action, has remained unclear. we report here a binding site for ppgpp on e. coli rnap, identified by crosslinking, protease mapping, and analysis of muta ...201323623682
zinc-binding and structural properties of the histidine-rich loop of arabidopsis thaliana vacuolar membrane zinc transporter mtp1.the vacuolar zn(2+)/h(+) antiporter of arabidopsis thaliana, atmtp1, has a cytosolic histidine-rich loop (his-loop). we characterized the structures and zn(2+)-binding properties of the his-loop and other domains. circular dichroism analyses revealed that the his-loop partly consists of a polyproline type ii structure and that its conformational change is induced by zn(2+) as well as the c-terminal domain. isothermal titration calorimetry of the his-loop revealed a binding number of four zn(2+) ...201323772397
structure of the protein core of translation initiation factor 2 in apo, gtp-bound and gdp-bound forms.translation initiation factor 2 (if2) is involved in the early steps of bacterial protein synthesis. it promotes the stabilization of the initiator trna on the 30s initiation complex (ic) and triggers gtp hydrolysis upon ribosomal subunit joining. while the structure of an archaeal homologue (a/eif5b) is known, there are significant sequence and functional differences in eubacterial if2, while the trimeric eukaryotic if2 is completely unrelated. here, the crystal structure of the apo if2 protein ...201323695237
structure of the archaeal cascade subunit csa5: relating the small subunits of crispr effector complexes.the cascade complex for crispr-mediated antiviral immunity uses crispr rna (crrna) to target invading dna species from mobile elements such as viruses, leading to their destruction. the core of the cascade effector complex consists of the cas5 and cas7 subunits, which are widely conserved in prokaryotes. cas7 binds crrna and forms the helical backbone of cascade. many archaea encode a version of the cascade complex (denoted type i-a) that includes a csa5 (or small) subunit, which interacts weakl ...201323846216
yidc occupies the lateral gate of the secyeg translocon and is sequentially displaced by a nascent membrane protein.most membrane proteins are co-translationally inserted into the lipid bilayer via the universally conserved secy complex and they access the lipid phase presumably via a lateral gate in secy. in bacteria, the lipid transfer of membrane proteins from the secy channel is assisted by the secy-associated protein yidc, but details on the secy-yidc interaction are unknown. by employing an in vivo and in vitro site-directed cross-linking approach, we have mapped the secy-yidc interface and found yidc i ...201323609445
an aromatic residue switch in enhancer-dependent bacterial rna polymerase controls transcription intermediate complex activity.the formation of the open promoter complex (rpo) in which the melted dna containing the transcription start site is located at the rna polymerase (rnap) catalytic centre is an obligatory step in the transcription of dna into rna catalyzed by rnap. in the rpo, an extensive network of interactions is established between dna, rnap and the σ-factor and the formation of functional rpo occurs via a series of transcriptional intermediates (collectively 'rpi'). a single tryptophan is ideally positioned ...201323609536
bacillus subtilis rna deprotection enzyme rpph recognizes guanosine in the second position of its substrates.the initiation of mrna degradation often requires deprotection of its 5' end. in eukaryotes, the 5'-methylguanosine (cap) structure is principally removed by the nudix family decapping enzyme dcp2, yielding a 5'-monophosphorylated rna that is a substrate for 5' exoribonucleases. in bacteria, the 5'-triphosphate group of primary transcripts is also converted to a 5' monophosphate by a nudix protein called rna pyrophosphohydrolase (rpph), allowing access to both endo- and 5' exoribonucleases. here ...201323610407
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