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determining structures of rna aptamers and riboswitches by x-ray crystallography.structural biology plays a central role in gaining a full understanding of the myriad roles of rna in biology. in recent years, innovative approaches in rna purification and crystallographic methods have lead to the visualization of an increasing number of unique structures, providing new insights into its function at the atomic level. this article presents general protocols which have streamlined the process of obtaining a homogeneous sample of properly folded and active rna in high concentrati ...200919377976
high-throughput haplotype determination over long distances by haplotype fusion pcr and ligation haplotyping.when combined with haplotype fusion pcr (hf-pcr), ligation haplotyping is a robust, high-throughput method for empirical determination of haplotypes, which can be applied to assaying both sequence and structural variation over long distances. unlike alternative approaches to haplotype determination, such as allele-specific pcr and long pcr, hf-pcr and ligation haplotyping do not suffer from mispriming or template-switching errors. in this method, hf-pcr is used to juxtapose dna sequences from si ...200920010928
lessons from structural genomics.a decade of structural genomics, the large-scale determination of protein structures, has generated a wealth of data and many important lessons for structural biology and for future large-scale projects. these lessons include a confirmation that it is possible to construct large-scale facilities that can determine the structures of a hundred or more proteins per year, that these structures can be of high quality, and that these structures can have an important impact. technology development has ...200919416074
pharmacological targeting of the hsp70 chaperone.the molecular chaperone, heat shock protein 70 (hsp70), acts at multiple steps in a protein's life cycle, including during the processes of folding, trafficking, remodeling and degradation. to accomplish these various tasks, the activity of hsp70 is shaped by a host of co-chaperones, which bind to the core chaperone and influence its functions. genetic studies have strongly linked hsp70 and its co-chaperones to numerous diseases, including cancer, neurodegeneration and microbial pathogenesis, ye ...200919860737
the biogenesis and function of piwi proteins and pirnas: progress and prospect.the evolutionarily conserved argonaute/piwi (ago/piwi, also known as paz-piwi domain or ppd) family of proteins is crucial for the biogenesis and function of small noncoding rnas (ncrnas). this family can be divided into ago and piwi subfamilies. the ago proteins are ubiquitously present in diverse tissues. they bind to small interfering rnas (sirnas) and micrornas (mirnas). in contrast, the piwi proteins are predominantly present in the germline and associate with a novel class of small rnas kn ...200919575643
form, function, and regulation of argonaute proteins.both transcriptional (tgs) and posttranscriptional gene silencing (ptgs) are conserved eukaryotic gene regulatory mechanisms, integral for taming exogenous (viruses and bacteria) or endogenous (repetitive elements and transposons) invasive nucleic acids to minimize their impact on genome integrity and function. tgs and ptgs also are essential for controlling the expression of protein coding genes throughout development or in response to environmental stimuli. in plants and animals, at least one ...201021183704
nanometer propagation of millisecond motions in v-type allostery.imidazole glycerol phosphate synthase (igps) is a v-type allosteric enzyme, which is catalytically inactive for glutamine hydrolysis until the allosteric effector, n'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (prfar) binds 30 å away. in the apo state, nmr relaxation dispersion experiments indicate the absence of millisecond (ms) timescale motions. binding of the prfar to form the active ternary complex is endothermic with a large positive entropy change. in a ...201021134639
phylogenomic analysis of the uracil-dna glycosylase superfamily.the spontaneous deamination of cytosine produces uracil mispaired with guanine in dna, which will produce a mutation, unless repaired. in all domains of life, uracil-dna glycosylases (udgs) are responsible for the elimination of uracil from dna. thus, udgs contribute to the integrity of the genetic information and their loss results in mutator phenotypes. we are interested in understanding the role of udg genes in the evolutionary variation of the rate and the spectrum of spontaneous mutations. ...201021135150
phylogenomic analysis of the uracil-dna glycosylase superfamily.the spontaneous deamination of cytosine produces uracil mispaired with guanine in dna, which will produce a mutation, unless repaired. in all domains of life, uracil-dna glycosylases (udgs) are responsible for the elimination of uracil from dna. thus, udgs contribute to the integrity of the genetic information and their loss results in mutator phenotypes. we are interested in understanding the role of udg genes in the evolutionary variation of the rate and the spectrum of spontaneous mutations. ...201021135150
the folding of single domain proteins--have we reached a consensus?rather than stressing the most recent advances in the field, this review highlights the fundamental topics where disagreement remains and where adequate experimental data are lacking. these topics include properties of the denatured state and the role of residual structure, the nature of the fundamental steps and barriers, the extent of pathway heterogeneity and non-native interactions, recent comparisons between theory and experiment, and finally, dynamical properties of the folding reaction.201021144739
the folding of single domain proteins--have we reached a consensus?rather than stressing the most recent advances in the field, this review highlights the fundamental topics where disagreement remains and where adequate experimental data are lacking. these topics include properties of the denatured state and the role of residual structure, the nature of the fundamental steps and barriers, the extent of pathway heterogeneity and non-native interactions, recent comparisons between theory and experiment, and finally, dynamical properties of the folding reaction.201021144739
small rna sorting: matchmaking for argonautes.small rnas directly or indirectly impact nearly every biological process in eukaryotic cells. to perform their myriad roles, not only must precise small rna species be generated, but they must also be loaded into specific effector complexes called rna-induced silencing complexes (riscs). argonaute proteins form the core of riscs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. in this review, we explore the mecha ...201021116305
small rna sorting: matchmaking for argonautes.small rnas directly or indirectly impact nearly every biological process in eukaryotic cells. to perform their myriad roles, not only must precise small rna species be generated, but they must also be loaded into specific effector complexes called rna-induced silencing complexes (riscs). argonaute proteins form the core of riscs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. in this review, we explore the mecha ...201021116305
the bridge helix coordinates movements of modules in rna polymerase.the rna polymerase 'bridge helix' is a metastable α-helix that spans the leading edge of the enzyme active-site cleft. a new study published in bmc biology reveals surprising tolerance to helix-disrupting changes in a region previously thought crucial for translocation, and suggests roles for two hinge-like segments of the bridge helix in coordinating modules that move during the nucleotide-addition cycle.201021114873
synonymous but not the same: the causes and consequences of codon bias.despite their name, synonymous mutations have significant consequences for cellular processes in all taxa. as a result, an understanding of codon bias is central to fields as diverse as molecular evolution and biotechnology. although recent advances in sequencing and synthetic biology have helped to resolve longstanding questions about codon bias, they have also uncovered striking patterns that suggest new hypotheses about protein synthesis. ongoing work to quantify the dynamics of initiation an ...201021102527
synonymous but not the same: the causes and consequences of codon bias.despite their name, synonymous mutations have significant consequences for cellular processes in all taxa. as a result, an understanding of codon bias is central to fields as diverse as molecular evolution and biotechnology. although recent advances in sequencing and synthetic biology have helped to resolve longstanding questions about codon bias, they have also uncovered striking patterns that suggest new hypotheses about protein synthesis. ongoing work to quantify the dynamics of initiation an ...201021102527
interaction of calmodulin with sec61α limits ca2+ leakage from the endoplasmic reticulum.in eukaryotes, protein transport into the endoplasmic reticulum (er) is facilitated by a protein-conducting channel, the sec61 complex. the presence of large, water-filled pores with uncontrolled ion permeability, as formed by sec61 complexes in the er membrane, would seriously interfere with the regulated release of calcium from the er lumen into the cytosol, an essential mechanism for intracellular signalling. we identified a calmodulin (cam)-binding motif in the cytosolic n-terminus of mammal ...201021102557
interaction of calmodulin with sec61α limits ca2+ leakage from the endoplasmic reticulum.in eukaryotes, protein transport into the endoplasmic reticulum (er) is facilitated by a protein-conducting channel, the sec61 complex. the presence of large, water-filled pores with uncontrolled ion permeability, as formed by sec61 complexes in the er membrane, would seriously interfere with the regulated release of calcium from the er lumen into the cytosol, an essential mechanism for intracellular signalling. we identified a calmodulin (cam)-binding motif in the cytosolic n-terminus of mammal ...201021102557
application of a high-throughput fluorescent acetyltransferase assay to identify inhibitors of homocitrate synthase.homocitrate synthase (hcs) catalyzes the first step of l-lysine biosynthesis in fungi by condensing acetyl-coenzyme a and 2-oxoglutarate to form 3r-homocitrate and coenzyme a. due to its conservation in pathogenic fungi, hcs has been proposed as a candidate for antifungal drug design. here we report the development and validation of a robust fluorescent assay for hcs that is amenable to high-throughput screening for inhibitors in vitro. using this assay, schizosaccharomyces pombe hcs was screene ...201021073853
application of a high-throughput fluorescent acetyltransferase assay to identify inhibitors of homocitrate synthase.homocitrate synthase (hcs) catalyzes the first step of l-lysine biosynthesis in fungi by condensing acetyl-coenzyme a and 2-oxoglutarate to form 3r-homocitrate and coenzyme a. due to its conservation in pathogenic fungi, hcs has been proposed as a candidate for antifungal drug design. here we report the development and validation of a robust fluorescent assay for hcs that is amenable to high-throughput screening for inhibitors in vitro. using this assay, schizosaccharomyces pombe hcs was screene ...201021073853
psortdb--an expanded, auto-updated, user-friendly protein subcellular localization database for bacteria and archaea.the subcellular localization (scl) of a microbial protein provides clues about its function, its suitability as a drug, vaccine or diagnostic target and aids experimental design. the first version of psortdb provided a valuable resource comprising a data set of proteins of known scl (epsortdb) as well as pre-computed scl predictions for proteomes derived from complete bacterial genomes (cpsortdb). psortdb 2.0 (http://db.psort.org) extends user-friendly functionalities, significantly expands epso ...201021071402
psortdb--an expanded, auto-updated, user-friendly protein subcellular localization database for bacteria and archaea.the subcellular localization (scl) of a microbial protein provides clues about its function, its suitability as a drug, vaccine or diagnostic target and aids experimental design. the first version of psortdb provided a valuable resource comprising a data set of proteins of known scl (epsortdb) as well as pre-computed scl predictions for proteomes derived from complete bacterial genomes (cpsortdb). psortdb 2.0 (http://db.psort.org) extends user-friendly functionalities, significantly expands epso ...201021071402
quantum chemical studies of nucleic acids: can we construct a bridge to the rna structural biology and bioinformatics communities?in this feature article, we provide a side-by-side introduction for two research fields: quantum chemical calculations of molecular interaction in nucleic acids and rna structural bioinformatics. our main aim is to demonstrate that these research areas, while largely separated in contemporary literature, have substantial potential to complement each other that could significantly contribute to our understanding of the exciting world of nucleic acids. we identify research questions amenable to th ...201021049899
structural basis for recognition of s-adenosylhomocysteine by riboswitches.s-adenosyl-(l)-homocysteine (sah) riboswitches are regulatory elements found in bacterial mrnas that up-regulate genes involved in the s-adenosyl-(l)-methionine (sam) regeneration cycle. to understand the structural basis of sah-dependent regulation by rna, we have solved the structure of its metabolite-binding domain in complex with sah. this structure reveals an unusual pseudoknot topology that creates a shallow groove on the surface of the rna that binds sah primarily through interactions wit ...201020864509
fluorescence-based assay to measure the real-time kinetics of nucleotide incorporation during transcription elongation.understanding the mechanism and fidelity of transcription by the rna polymerase (rnap) requires measurement of the dissociation constant (k(d)) of correct and incorrect ntps and their incorporation rate constants (k(pol)). currently, such parameters are obtained from radiometric-based assays that are both tedious and discontinuous. here, we report a fluorescence-based assay for measuring the real-time kinetics of single-nucleotide incorporation during transcription elongation. the fluorescent ad ...201021035457
fluorescence-based assay to measure the real-time kinetics of nucleotide incorporation during transcription elongation.understanding the mechanism and fidelity of transcription by the rna polymerase (rnap) requires measurement of the dissociation constant (k(d)) of correct and incorrect ntps and their incorporation rate constants (k(pol)). currently, such parameters are obtained from radiometric-based assays that are both tedious and discontinuous. here, we report a fluorescence-based assay for measuring the real-time kinetics of single-nucleotide incorporation during transcription elongation. the fluorescent ad ...201021035457
integrated prediction of one-dimensional structural features and their relationships with conformational flexibility in helical membrane proteins.many structural properties such as solvent accessibility, dihedral angles and helix-helix contacts can be assigned to each residue in a membrane protein. independent studies exist on the analysis and sequence-based prediction of some of these so-called one-dimensional features. however, there is little explanation of why certain residues are predicted in a wrong structural class or with large errors in the absolute values of these features. on the other hand, membrane proteins undergo conformati ...201020977780
phosphate binding sites identification in protein structures.nearly half of known protein structures interact with phosphate-containing ligands, such as nucleotides and other cofactors. many methods have been developed for the identification of metal ions-binding sites and some for bigger ligands such as carbohydrates, but none is yet available for the prediction of phosphate-binding sites. here we describe pfinder, a method that predicts binding sites for phosphate groups, both in the form of ions or as parts of other non-peptide ligands, in proteins of ...201020974634
phosphate binding sites identification in protein structures.nearly half of known protein structures interact with phosphate-containing ligands, such as nucleotides and other cofactors. many methods have been developed for the identification of metal ions-binding sites and some for bigger ligands such as carbohydrates, but none is yet available for the prediction of phosphate-binding sites. here we describe pfinder, a method that predicts binding sites for phosphate groups, both in the form of ions or as parts of other non-peptide ligands, in proteins of ...201020974634
electron tunneling in respiratory complex i.nadh:ubiquinone oxidoreductase (complex i) plays a central role in the respiratory electron transport chain by coupling the transfer of electrons from nadh to ubiquinone to the creation of the proton gradient across the membrane necessary for atp synthesis. here the atomistic details of electronic wiring of all fe/s clusters in complex i are revealed by using the tunneling current theory and computer simulations; both density functional theory and semiempirical electronic structure methods were ...201020974925
r3d align: global pairwise alignment of rna 3d structures using local superpositions.comparing 3d structures of homologous rna molecules yields information about sequence and structural variability. to compare large rna 3d structures, accurate automatic comparison tools are needed. in this article, we introduce a new algorithm and web server to align large homologous rna structures nucleotide by nucleotide using local superpositions that accommodate the flexibility of rna molecules. local alignments are merged to form a global alignment by employing a maximum clique algorithm on ...201020929913
an arabidopsis pentatricopeptide repeat protein, suppressor of variegation7, is required for ftsh-mediated chloroplast biogenesis.the arabidopsis (arabidopsis thaliana) yellow variegated2 (var2) mutant has green- and white-sectored leaves due to loss of var2, a subunit of the chloroplast ftsh protease/chaperone complex. suppressor screens are a valuable tool to gain insight into var2 function and the mechanism of var2 variegation. here, we report the molecular characterization of 004-003, a line in which var2 variegation is suppressed. we found that the suppression phenotype in this line is caused by lack of a chloroplast ...201020935174
promoting rna helical stacking via a-minor junctions.rna molecules take advantage of prevalent structural motifs to fold and assemble into well-defined 3d architectures. the a-minor junction is a class of rna motifs that specifically controls coaxial stacking of helices in natural rnas. a sensitive self-assembling supra-molecular system was used as an assay to compare several natural and previously unidentified a-minor junctions by native polyacrylamide gel electrophoresis and atomic force microscopy. this class of modular motifs follows a topolog ...201020876687
promoting rna helical stacking via a-minor junctions.rna molecules take advantage of prevalent structural motifs to fold and assemble into well-defined 3d architectures. the a-minor junction is a class of rna motifs that specifically controls coaxial stacking of helices in natural rnas. a sensitive self-assembling supra-molecular system was used as an assay to compare several natural and previously unidentified a-minor junctions by native polyacrylamide gel electrophoresis and atomic force microscopy. this class of modular motifs follows a topolog ...201020876687
designer drugs for discerning bugs. 201020876111
fungal laccases: production, function, and applications in food processing.laccases are increasingly being used in food industry for production of cost-effective and healthy foods. to sustain this trend widespread availability of laccase and efficient production systems have to be developed. the present paper delineate the recent developments that have taken place in understanding the role of laccase action, efforts in overexpression of laccase in heterologous systems, and various cultivation techniques that have been developed to efficiently produce laccase at the ind ...201021048859
comparative study of three commonly used continuous deterministic methods for modeling gene regulation networks.a gene-regulatory network (grn) refers to dna segments that interact through their rna and protein products and thereby govern the rates at which genes are transcribed. creating accurate dynamic models of grns is gaining importance in biomedical research and development. to improve our understanding of continuous deterministic modeling methods employed to construct dynamic grn models, we have carried out a comprehensive comparative study of three commonly used systems of ordinary differential eq ...201020840745
preparation and high-resolution microscopy of gold cluster labeled nucleic acid conjugates and nanodevices.nanogold and undecagold are covalently linked gold cluster labels which enable the identification and localization of biological components with molecular precision and resolution. they can be prepared with different reactivities, which means they can be conjugated to a wide variety of molecules, including nucleic acids, at specific, unique sites. the location of these sites can be synthetically programmed in order to preserve the binding affinity of the conjugate and impart novel characteristic ...201020869258
preparation and high-resolution microscopy of gold cluster labeled nucleic acid conjugates and nanodevices.nanogold and undecagold are covalently linked gold cluster labels which enable the identification and localization of biological components with molecular precision and resolution. they can be prepared with different reactivities, which means they can be conjugated to a wide variety of molecules, including nucleic acids, at specific, unique sites. the location of these sites can be synthetically programmed in order to preserve the binding affinity of the conjugate and impart novel characteristic ...201020869258
fidelity in archaeal information processing.a key element during the flow of genetic information in living systems is fidelity. the accuracy of dna replication influences the genome size as well as the rate of genome evolution. the large amount of energy invested in gene expression implies that fidelity plays a major role in fitness. on the other hand, an increase in fidelity generally coincides with a decrease in velocity. hence, an important determinant of the evolution of life has been the establishment of a delicate balance between fi ...201020871851
mathematics, thermodynamics, and modeling to address ten common misconceptions about protein structure, folding, and stability.to fully understand the roles proteins play in cellular processes, students need to grasp complex ideas about protein structure, folding, and stability. our current understanding of these topics is based on mathematical models and experimental data. however, protein structure, folding, and stability are often introduced as descriptive, qualitative phenomena in undergraduate classes. in the process of learning about these topics, students often form incorrect ideas. for example, by learning about ...201020810950
origin and evolution of the ribosome.the modern ribosome was largely formed at the time of the last common ancestor, luca. hence its earliest origins likely lie in the rna world. central to its development were rnas that spawned the modern trnas and a symmetrical region deep within the large ribosomal rna, (rrna), where the peptidyl transferase reaction occurs. to understand pre-luca developments, it is argued that events that are coupled in time are especially useful if one can infer a likely order in which they occurred. using su ...201020534711
friedreich ataxia: molecular mechanisms, redox considerations, and therapeutic opportunities.mitochondrial dysfunction and oxidative damage are at the origin of numerous neurodegenerative diseases like friedreich ataxia and alzheimer and parkinson diseases. friedreich ataxia (frda) is the most common hereditary ataxia, with one individual affected in 50,000. this disease is characterized by progressive degeneration of the central and peripheral nervous systems, cardiomyopathy, and increased incidence of diabetes mellitus. frda is caused by a dynamic mutation, a gaa trinucleotide repeat ...201020156111
rnamotifscan: automatic identification of rna structural motifs using secondary structural alignment.recent studies have shown that rna structural motifs play essential roles in rna folding and interaction with other molecules. computational identification and analysis of rna structural motifs remains a challenging task. existing motif identification methods based on 3d structure may not properly compare motifs with high structural variations. other structural motif identification methods consider only nested canonical base-pairing structures and cannot be used to identify complex rna structura ...201020696653
the role of l1 stalk-trna interaction in the ribosome elongation cycle.the ribosomal l1 stalk is a mobile structure implicated in directing trna movement during translocation through the ribosome. this article investigates three aspects of l1 stalk-trna interaction. first, by combining data from cryo electron microscopy, x-ray crystallography, and molecular dynamics simulations through the molecular dynamics flexible fitting method, we obtained atomic models of different trnas occupying the hybrid p/e state interacting with the l1 stalk. these models confirm the as ...201020691699
a structural database for k-turn motifs in rna.the kink-turn (k-turn) is a common structural motif in rna that introduces a tight kink into the helical axis. k-turns play an important architectural role in rna structures and serve as binding sites for a number of proteins. we have created a database of known and postulated k-turn sequences and three-dimensional (3d) structures, available via the internet. this site provides (1) a database of sequence and structure, as a resource for the rna community, and (2) a tool to enable the manipulatio ...201020562215
synchrotron x-ray-induced photoreduction of ferric myoglobin nitrite crystals gives the ferrous derivative with retention of the o-bonded nitrite ligand.exposure of a single crystal of the nitrite adduct of ferric myoglobin (mb) at 100 k to high-intensity synchrotron x-ray radiation resulted in changes in the uv-vis spectrum that can be attributed to reduction of the ferric compound to the ferrous derivative. we employed correlated single-crystal spectroscopy with crystallography to further characterize this photoproduct. the 1.55 a resolution crystal structure of the photoproduct reveals retention of the o-binding mode for binding of nitrite to ...201020568729
nmr structure of the let-7 mirna interacting with the site lcs1 of lin-41 mrna from caenorhabditis elegans.we have determined the 3d structure of a 34-nt rna construct, herein named lcs1co, which mimics the interaction of let-7 microrna (mirna) to one of its complementary binding sites, lcs1, in the 3'-untranslated region of lin-41 mrna by solution-state nmr spectroscopy. let-7 mirnas control the timing of development of the nematode caenorhabditis elegans and are highly conserved in mammals. the sequence and structure of the two conserved let-7 complementary sites, lcs1 and lcs2, in the 3'-untransla ...201020660479
a molecular clamp ensures allosteric coordination of peptidyltransfer and ligand binding to the ribosomal a-site.although the ribosome is mainly comprised of rrna and many of its critical functions occur through rna-rna interactions, distinct domains of ribosomal proteins also participate in switching the ribosome between different conformational/functional states. prior studies demonstrated that two extended domains of ribosomal protein l3 form an allosteric switch between the pre- and post-translocational states. missing was an explanation for how the movements of these domains are communicated among the ...201020660012
bioinspired heme, heme/nonheme diiron, heme/copper, and inorganic nox chemistry: *no((g)) oxidation, peroxynitrite-metal chemistry, and *no((g)) reductive coupling.the focus of this forum article highlights work from our own laboratories and those of others in the area of biochemical and biologically inspired inorganic chemistry dealing with nitric oxide [nitrogen monoxide, *no((g))] and its biological roles and reactions. the latter focus is on (i) oxidation of *no((g)) to nitrate by nitric oxide dioxygenases (nods) and (ii) reductive coupling of two molecules of *no((g)) to give n(2)o(g). in the former case, nods are described, and the highlighting of po ...201020666386
a polyhedron made of trnas.supramolecular assembly is a powerful strategy used by nature to build nanoscale architectures with predefined sizes and shapes. with synthetic systems, however, numerous challenges remain to be solved before precise control over the synthesis, folding and assembly of rationally designed three-dimensional nano-objects made of rna can be achieved. here, using the transfer rna molecule as a structural building block, we report the design, efficient synthesis and structural characterization of stab ...201020729899
electron cryomicroscopy of membrane proteins: specimen preparation for two-dimensional crystals and single particles.membrane protein structure and function can be studied by two powerful and highly complementary electron cryomicroscopy (cryo-em) methods: electron crystallography of two-dimensional (2d) crystals and single particle analysis of detergent-solubilized protein complexes. to obtain the highest-possible resolution data from membrane proteins, whether prepared as 2d crystals or single particles, cryo-em samples must be vitrified with great care. grid preparation for cryo-em of 2d crystals is possible ...201020678942
electron cryomicroscopy of membrane proteins: specimen preparation for two-dimensional crystals and single particles.membrane protein structure and function can be studied by two powerful and highly complementary electron cryomicroscopy (cryo-em) methods: electron crystallography of two-dimensional (2d) crystals and single particle analysis of detergent-solubilized protein complexes. to obtain the highest-possible resolution data from membrane proteins, whether prepared as 2d crystals or single particles, cryo-em samples must be vitrified with great care. grid preparation for cryo-em of 2d crystals is possible ...201020678942
definition and estimation of resolution in single-particle reconstructions.in this paper, we review current practices for establishing the resolution in single-particle reconstructions. the classical raleigh criterion for the resolution is not applicable in this case, and the resolution is commonly defined by a consistency test, whereby the data set is randomly split in half and the two resulting reconstructions are then compared. such a procedure, however, may introduce statistical dependence between the two half-sets, which leads to a too optimistic resolution estima ...201020637413
glycosidic bond conformation preference plays a pivotal role in catalysis of rna pseudouridylation: a combined simulation and structural study.the most abundant chemical modification on rna is isomerization of uridine (or pseudouridylation) catalyzed by pseudouridine synthases. the catalytic mechanism of this essential process remains largely speculative, partly due to lack of knowledge of the pre-reactive state that is important to the identification of reactive chemical moieties. in the present study, we showed, using orthogonal space random-walk free-energy simulation, that the pre-reactive states of uridine and its reactive derivat ...201020615421
automatic structure classification of small proteins using random forest.random forest, an ensemble based supervised machine learning algorithm, is used to predict the scop structural classification for a target structure, based on the similarity of its structural descriptors to those of a template structure with an equal number of secondary structure elements (sses). an initial assessment of random forest is carried out for domains consisting of three sses. the usability of random forest in classifying larger domains is demonstrated by applying it to domains consist ...201020594334
structural aspects of phenylalanylation and quality control in three major forms of phenylalanyl-trna synthetase.aminoacyl-trna synthetases (aarss) are a canonical set of enzymes that specifically attach corresponding amino acids to their cognate transfer rnas in the cytoplasm, mitochondria, and nucleus. the aarss display great differences in primary sequence, subunit size, and quaternary structure. existence of three types of phenylalanyl-trna synthetase (phers)-bacterial (αβ)(2), eukaryotic/archaeal cytosolic (αβ)(2), and mitochondrial α-is a prominent example of structural diversity within the aarss fam ...201022331999
the transcription inhibitor lipiarmycin blocks dna fitting into the rna polymerase catalytic site.worldwide spreading of drug-resistant pathogens makes mechanistic understanding of antibiotic action an urgent task. the macrocyclic antibiotic lipiarmycin (lpm), which is under development for clinical use, inhibits bacterial rna polymerase (rnap) by an unknown mechanism. using genetic and biochemical approaches, we show that lpm targets the sigma(70) subunit region 3.2 and the rnap beta' subunit switch-2 element, which controls the clamping of promoter dna in the rnap active-site cleft. lpm ab ...201020562828
quantifying the relationship between sequence and three-dimensional structure conservation in rna.in recent years, the number of available rna structures has rapidly grown reflecting the increased interest on rna biology. similarly to the studies carried out two decades ago for proteins, which gave the fundamental grounds for developing comparative protein structure prediction methods, we are now able to quantify the relationship between sequence and structure conservation in rna.201020550657
crystal structure and ligand binding of the mid domain of a eukaryotic argonaute protein.argonaute (ago) proteins are core components of rna-induced silencing complexes and have essential roles in rna-mediated gene silencing. they are characterized by a bilobal architecture, consisting of one lobe containing the amino-terminal and paz domains and another containing the mid and piwi domains. except for the paz domain, structural information on eukaryotic ago domains is not yet available. in this study, we report the crystal structure of the mid domain of the eukaryotic ago protein qd ...201020539312
metal ion roles and the movement of hydrogen during reaction catalyzed by d-xylose isomerase: a joint x-ray and neutron diffraction study.conversion of aldo to keto sugars by the metalloenzyme d-xylose isomerase (xi) is a multistep reaction that involves hydrogen transfer. we have determined the structure of this enzyme by neutron diffraction in order to locate h atoms (or their isotope d). two studies are presented, one of xi containing cadmium and cyclic d-glucose (before sugar ring opening has occurred), and the other containing nickel and linear d-glucose (after ring opening has occurred but before isomerization). previously w ...201020541506
all things must pass: contrasts and commonalities in eukaryotic and bacterial mrna decay.despite its universal importance for controlling gene expression, mrna degradation was initially thought to occur by disparate mechanisms in eukaryotes and bacteria. this conclusion was based on differences in the structures used by these organisms to protect mrna termini and in the rnases and modifying enzymes originally implicated in mrna decay. subsequent discoveries have identified several striking parallels between the cellular factors and molecular events that govern mrna degradation in th ...201020520623
location of macular xanthophylls in the most vulnerable regions of photoreceptor outer-segment membranes.lutein and zeaxanthin are two dietary carotenoids that compose the macular pigment of the primate retina. another carotenoid, meso-zeaxanthin, is formed from lutein in the retina. a membrane location is one possible site where these dipolar, terminally dihydroxylated carotenoids, named macular xanthophylls, are accumulated in the nerve fibers and photoreceptor outer segments. macular xanthophylls are oriented perpendicular to the membrane surface, which ensures their high solubility, stability, ...201020494651
a novel atp-dependent conformation in p97 n-d1 fragment revealed by crystal structures of disease-related mutants.mutations in p97, a major cytosolic aaa (atpases associated with a variety of cellular activities) chaperone, cause inclusion body myopathy associated with paget's disease of the bone and frontotemporal dementia (ibmpfd). ibmpfd mutants have single amino-acid substitutions at the interface between the n-terminal domain (n-domain) and the adjacent aaa domain (d1), resulting in a reduced affinity for adp. the structures of p97 n-d1 fragments bearing ibmpfd mutations adopt an atypical n-domain conf ...201020512113
iparts: an improved tool of pairwise alignment of rna tertiary structures.iparts is an improved web server for aligning two rna 3d structures based on a structural alphabet (sa)-based approach. in particular, we first derive a ramachandran-like diagram of rnas by plotting nucleotides on a 2d axis using their two pseudo-torsion angles eta and . next, we apply the affinity propagation clustering algorithm to this eta- plot to obtain an sa of 23-nt conformations. we finally use this sa to transform rna 3d structures into 1d sequences of sa letters and continue to utilize ...201020507908
alkyltransferase-like proteins: molecular switches between dna repair pathways.alkyltransferase-like proteins (atls) play a role in the protection of cells from the biological effects of dna alkylation damage. although atls share functional motifs with the dna repair protein and cancer chemotherapy target o⁶-alkylguanine-dna alkyltransferase, they lack the reactive cysteine residue required for alkyltransferase activity, so its mechanism for cell protection was previously unknown. here we review recent advances in unraveling the enigmatic cellular protection provided by at ...201020502938
structural signatures of antibiotic binding sites on the ribosome.the ribosome represents a major target for antibacterial drugs. being a complex molecular machine, it offers many potential sites for functional interference. the high-resolution structures of ribosome in complex with various antibiotics provide a unique data set for understanding the universal features of drug-binding pockets on the ribosome. in this work, we have analyzed the structural and evolutionary properties of 65 antibiotic binding sites (abss) in the ribosome. we compared these sites t ...201020494981
a'-form rna helices are required for cytoplasmic mrna transport in drosophila.microtubule-based mrna transport is widely used to restrict protein expression to specific regions in the cell and has important roles in defining cell polarity and axis determination as well as in neuronal function. however, the structural basis of recognition of cis-acting mrna localization signals by motor complexes is poorly understood. we have used nmr spectroscopy to describe the first tertiary structure to our knowledge of an rna element responsible for mrna transport. the drosophila mela ...201020473315
the crystal structure of dynein intermediate chain-light chain roadblock complex gives new insights into dynein assembly.the roadblock/lc7 dynein light chain is a ubiquitous component of all dyneins and is essential for many diverse processes including proper axonal transport and dendrite growth. in addition, lc7 functions in non-dynein transcriptional activation of the transforming growth factor-beta complex. crystal structures of drosophila melanogaster lc7 in the apo form and in complex with a segment of the disordered n-terminal domain of dynein intermediate chain (ic) provide the first definitive identificati ...201020472935
invariant u2 snrna nucleotides form a stem loop to recognize the intron early in splicing.u2 snrna-intron branchpoint pairing is a critical step in pre-mrna recognition by the splicing apparatus, but the mechanism by which these two rnas engage each other is unknown. here, we identify a u2 snrna structure, the branchpoint-interacting stem loop (bsl), which presents the u2 nucleotides that will contact the intron. we provide evidence that the bsl forms prior to interaction with the intron and is disrupted by the dexd/h protein prp5p during engagement of the snrna with the intron. in v ...201020471947
nanosecond time-resolved polarization spectroscopies: tools for probing protein reaction mechanisms.polarization methods, introduced in the 1800s, offered one of the earliest ways to examine protein structure. since then, many other structure-sensitive probes have been developed, but circular dichroism (cd) remains a powerful technique because of its versatility and the specificity of protein structural information that can be explored. with improvements in time resolution, from millisecond to picosecond cd measurements, it has proven to be an important tool for studying the mechanism of foldi ...201020438842
temperature adaptation at homologous sites in proteins from nine thermophile-mesophile species pairs.whether particular amino acids are favored by selection at high temperatures over others has long been an open question in protein evolution. one way to approach this question is to compare homologous sites in proteins from one thermophile and a closely related mesophile; asymmetrical substitution patterns have been taken as evidence for selection favoring certain amino acids over others. however, most pairs of prokaryotic species that differ in optimum temperature also differ in genome-wide gc ...201020624731
a screen of chemical modifications identifies position-specific modification by una to most potently reduce sirna off-target effects.small interfering rnas (sirnas) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent mirna-like behavior. such off-target effects are primarily mediated by the sequence-specific interaction between the sirna seed regions (position 2-8 of either sirna strand counting from the 5'-end) and complementary sequences in the 3'utr of (off-) targets. it was previously shown that chemical modification of sirnas ca ...201020453030
sbspks: structure based sequence analysis of polyketide synthases.polyketide synthases (pkss) catalyze biosynthesis of a diverse family of pharmaceutically important secondary metabolites. bioinformatics analysis of sequence and structural features of pks proteins plays a crucial role in discovery of new natural products by genome mining, as well as in design of novel secondary metabolites by biosynthetic engineering. the availability of the crystal structures of various pks catalytic and docking domains, and mammalian fatty acid synthase module prompted us to ...201020444870
structure and function of nematode rna-binding proteins.rna-binding proteins are critical effectors of gene expression. they guide mrna localization, translation, and stability, and potentially play a role in regulating mrna synthesis. the structural basis for rna recognition by rna-binding proteins is the key to understand how they target specific transcripts for regulation. compared to other metazoans, nematode genomes contain a significant expansion in several rna-binding protein families, including pumilio-fbf (puf), ttp-like zinc finger (tzf), a ...201020418095
biocrystallography: past, present, future.the evolution of biocrystallography from the pioneers' time to the present era of global biology is presented in relation to the development of methodological and instrumental advances for molecular sample preparation and structure elucidation over the last 6 decades. the interdisciplinarity of the field that generated cross-fertilization between physics- and biology-focused themes is emphasized. in particular, strategies to circumvent the main bottlenecks of biocrystallography are discussed. th ...201021119764
sf1 and sf2 helicases: family matters.helicases of the superfamily (sf) 1 and 2 are involved in virtually all aspects of rna and dna metabolism. sf1 and sf2 helicases share a catalytic core with high structural similarity, but different enzymes even within each sf perform a wide spectrum of distinct functions on diverse substrates. to rationalize similarities and differences between these helicases, we outline a classification based on protein families that are characterized by typical sequence, structural, and mechanistic features. ...201020456941
imprints of the genetic code in the ribosome.the establishment of the genetic code remains elusive nearly five decades after the code was elucidated. the stereochemical hypothesis postulates that the code developed from interactions between nucleotides and amino acids, yet supporting evidence in a biological context is lacking. we show here that anticodons are selectively enriched near their respective amino acids in the ribosome, and that such enrichment is significantly correlated with the canonical code over random codes. ribosomal anti ...201020385807
structural basis of transmembrane domain interactions in integrin signaling.cell surface receptors of the integrin family are pivotal to cell adhesion and migration. the activation state of heterodimeric alphabeta integrins is correlated to the association state of the single-pass alpha and beta transmembrane domains. the association of integrin alphaiibbeta3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (alphaiib) and tilted (beta3) helix relative to the membrane in congruence to the dissociated s ...201020168080
differences in the structure and dynamics of the apo- and palmitate-ligated forms of aedes aegypti sterol carrier protein 2 (aescp-2).sterol carrier protein-2 (scp-2) is a nonspecific lipid-binding protein expressed ubiquitously in most organisms. knockdown of scp-2 expression in mosquitoes has been shown to result in high mortality in developing adults and significantly lowered fertility. thus, it is of interest to determine the structure of mosquito scp-2 and to identify its mechanism of lipid binding. we report here high quality three-dimensional solution structures of scp-2 from aedes aegypti determined by nmr spectroscopy ...201020356842
riding in silence: a little snowboarding, a lot of small rnas.the recent symposium, rna silencing: mechanism, biology and applications, organized by phillip d. zamore (university of massachusetts medical school) and beverly davidson (university of iowa), and held in keystone, colorado, brought together scientists working on diverse aspects of rna silencing, a field that comprises a multitude of gene regulatory pathways guided by micrornas, small interfering rnas and piwi-interacting rnas.201020230614
determination of ensemble-average pairwise root mean-square deviation from experimental b-factors.root mean-square deviation (rmsd) after roto-translational least-squares fitting is a measure of global structural similarity of macromolecules used commonly. on the other hand, experimental x-ray b-factors are used frequently to study local structural heterogeneity and dynamics in macromolecules by providing direct information about root mean-square fluctuations (rmsf) that can also be calculated from molecular dynamics simulations. we provide a mathematical derivation showing that, given a set ...201020197040
internal architecture of mitochondrial complex i from arabidopsis thaliana.the nadh dehydrogenase complex (complex i) of the respiratory chain has unique features in plants. it is the main entrance site for electrons into the respiratory electron transfer chain, has a role in maintaining the redox balance of the entire plant cell and additionally comprises enzymatic side activities essential for other metabolic pathways. here, we present a proteomic investigation to elucidate its internal structure. arabidopsis thaliana complex i was purified by a gentle biochemical pr ...201020197505
aminoglycoside activity observed on single pre-translocation ribosome complexes. 201020154669
detection of novel recombinases in bacteriophage genomes unveils rad52, rad51 and gp2.5 remote homologs.homologous recombination is a key in contributing to bacteriophages genome repair, circularization and replication. no less than six kinds of recombinase genes have been reported so far in bacteriophage genomes, two (uvsx and gp2.5) from virulent, and four (sak, red beta, erf and sak4) from temperate phages. using profile-profile comparisons, structure-based modelling and gene-context analyses, we provide new views on the global landscape of recombinases in 465 bacteriophages. we show that sak, ...201020194117
fluctuations in biological and bioinspired electron-transfer reactions.central to theories of electron transfer (et) is the idea that nuclear motion generates a transition state that enables electron flow to proceed, but nuclear motion also induces fluctuations in the donor-acceptor (da) electronic coupling that is the rate-limiting parameter for nonadiabatic et. the interplay between the da energy gap and da coupling fluctuations is particularly noteworthy in biological et, where flexible protein and mobile water bridges take center stage. here, we discuss the cri ...201020192814
cell-specific differences in the requirements for translation quality control.protein synthesis has an overall error rate of approximately 10(-4) for each mrna codon translated. the fidelity of translation is mainly determined by two events: synthesis of cognate amino acid:trna pairs by aminoacyl-trna synthetases (aarss) and accurate selection of aminoacyl-trnas (aa-trnas) by the ribosome. to ensure faithful aa-trna synthesis, many aarss employ a proofreading ("editing") activity, such as phenylalanyl-trna synthetases (phers) that hydrolyze mischarged tyr-trna(phe). eukar ...201020160120
reductive coupling of nitrogen monoxide (*no) facilitated by heme/copper complexes.the interactions of nitrogen monoxide (*no; nitric oxide) with transition metal centers continue to be of great interest, in part due to their importance in biochemical processes. here, we describe *no((g)) reductive coupling chemistry of possible relevance to that process (i.e., nitric oxide reductase (nor) biochemistry), which occurs at the heme/cu active site of cytochrome c oxidases (ccos). in this report, heme/cu/*no((g)) activity is studied using 1:1 ratios of heme and copper complex compo ...201020030370
protein vivisection reveals elusive intermediates in folding.although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. here, we outline a powerful strategy to populate partially unfolded intermediates: a buried aliphatic residue is substituted with a charged residue (e.g., leu-->glu(-)) to destabilize and unfold a specific region of the protein. we applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the beta5-strand is unfolded. the intermediate ref ...201020144618
thermodynamic characterization of naturally occurring rna tetraloops.although tetraloops are one of the most frequently occurring secondary structure motifs in rna, less than one-third of the 30 most frequently occurring rna tetraloops have been thermodynamically characterized. therefore, 24 stem-loop sequences containing common tetraloops were optically melted, and the thermodynamic parameters deltah degrees , deltas degrees , deltag degrees (37,) and t(m) for each stem-loop were determined. these new experimental values, on average, are 0.7 kcal/mol different f ...201020047989
genome scale prediction of substrate specificity for acyl adenylate superfamily of enzymes based on active site residue profiles.enzymes belonging to acyl:coa synthetase (acs) superfamily activate wide variety of substrates and play major role in increasing the structural and functional diversity of various secondary metabolites in microbes and plants. however, due to the large sequence divergence within the superfamily, it is difficult to predict their substrate preference by annotation transfer from the closest homolog. therefore, a large number of acs sequences present in public databases lack any functional annotation ...201020105319
heterologous laccase production and its role in industrial applications.laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. these enzymes are implicated in a variety of biological activities. most of the laccases studied thus far are of fungal origin. the large range of substrates oxidized by laccases has raised interest in using them within different industrial fields, such as pulp delignification, textile dye bleaching, and bioremediation. laccases secret ...201021327057
the balance between pre- and post-transfer editing in trna synthetases.the fidelity of trna aminoacylation is dependent in part on amino acid editing mechanisms. a hydrolytic activity that clears mischarged trnas typically resides in an active site on the trna synthetase that is distinct from its synthetic aminoacylation active site. a second pre-transfer editing pathway that hydrolyzes the trna synthetase aminoacyl adenylate intermediate can also be activated. pre- and post-transfer editing activities can co-exist within a single trna synthetase resulting in a red ...201019941860
trnas: cellular barcodes for amino acids.the role of trna in translating the genetic code has received considerable attention over the last 50 years, and we now know in great detail how particular amino acids are specifically selected and brought to the ribosome in response to the corresponding mrna codon. over the same period, it has also become increasingly clear that the ribosome is not the only destination to which trnas deliver amino acids, with processes ranging from lipid modification to antibiotic biosynthesis all using aminoac ...201019903480
structural insights into rna interference.virtually all animals and plants utilize small rna molecules to control protein expression during different developmental stages and in response to viral infection. structural and mechanistic studies have begun to illuminate three fundamental aspects of these pathways: small rna biogenesis, formation of rna-induced silencing complexes (riscs), and targeting of complementary mrnas. here we review exciting recent progress in understanding how regulatory rnas are produced and how they trigger speci ...201020053548
fundamentals of three-dimensional reconstruction from projections.three-dimensional (3d) reconstruction of an object mass density from the set of its 2d line projections lies at a core of both single-particle reconstruction technique and electron tomography. both techniques utilize electron microscope to collect a set of projections of either multiple objects representing in principle the same macromolecular complex in an isolated form, or a subcellular structure isolated in situ. therefore, the goal of macromolecular electron microscopy is to invert the proje ...201020888956
trinuclear metal clusters in catalysis by terpenoid synthases.terpenoid synthases are ubiquitous enzymes that catalyze the formation of structurally and stereochemically diverse isoprenoid natural products. many isoprenoid coupling enzymes and terpenoid cyclases from bacteria, fungi, protists, plants, and animals share the class i terpenoid synthase fold. despite generally low amino acid sequence identity among these examples, class i terpenoid synthases contain conserved metal binding motifs that coordinate to a trinuclear metal cluster. this cluster not ...201021562622
an rna molecular switch: intrinsic flexibility of 23s rrna helices 40 and 68 5'-uaa/5'-gan internal loops studied by molecular dynamics methods.functional rna molecules such as ribosomal rnas frequently contain highly conserved internal loops with a 5'-uaa/5'-gan (uaa/gan) consensus sequence. the uaa/gan internal loops adopt distinctive structure inconsistent with secondary structure predictions. the structure has a narrow major groove and forms a trans hoogsteen/sugar edge (ths) a/g base pair followed by an unpaired stacked adenine, a trans watson-crick/hoogsteen (twh) u/a base pair and finally by a bulged nucleotide (n). the structure ...201021132104
the structure of eukaryotic and prokaryotic complex i.the structures of the nadh dehydrogenases from bos taurus and aquifex aeolicus have been determined by 3d electron microscopy, and have been analyzed in comparison with the previously determined structure of complex i from yarrowia lipolytica. the results show a clearly preserved domain structure in the peripheral arm of complex i, which is similar in the bacterial and eukaryotic complex. the membrane arms of both eukaryotic complexes show a similar shape but also significant differences in dist ...201019732833
the structure of eukaryotic and prokaryotic complex i.the structures of the nadh dehydrogenases from bos taurus and aquifex aeolicus have been determined by 3d electron microscopy, and have been analyzed in comparison with the previously determined structure of complex i from yarrowia lipolytica. the results show a clearly preserved domain structure in the peripheral arm of complex i, which is similar in the bacterial and eukaryotic complex. the membrane arms of both eukaryotic complexes show a similar shape but also significant differences in dist ...201019732833
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