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utilizing natural diversity to evolve protein function: applications towards thermostability.protein evolution relies on designing a library of sequences that capture meaningful functional diversity in a limited number of protein variants. several approaches take advantage of the sequence space already explored through natural selection by incorporating sequence diversity available from modern genomes (and their ancestors) when designing these libraries. the success of these approaches is, partly, owing to the fact that modern sequence diversity has already been subjected to evolutionar ...201121470898
an src homology 3 domain-like fold protein forms a ferredoxin binding site for the chloroplast nadh dehydrogenase-like complex in arabidopsis.some subunits of chloroplast nad(p)h dehydrogenase (ndh) are related to those of the respiratory complex i, and ndh mediates photosystem i (psi) cyclic electron flow. despite extensive surveys, the electron donor and its binding subunits have not been identified. here, we identified three novel components required for ndh activity. crrj and crrl are j- and j-like proteins, respectively, and are components of ndh subcomplex a. crr31 is an src homology 3 domain-like fold protein, and its c-termina ...201121505067
structural, mechanistic and coordination chemistry of relevance to the biosynthesis of iron-sulfur and related iron cofactors.iron-sulfur clusters are an important class of protein-bound prosthetic center that find wide utility in nature. roles include electron transfer, enzyme catalysis, protein structure stabilization, and regulation of gene expression as transcriptional and translational sensors. in eukaryotes their biosynthesis requires a complex molecular machinery that is located within the mitochondrion, while bacteria exhibit up to three independent cluster assembly pathways. all of these paths share common the ...201121499539
the genetic architecture of branched-chain amino acid accumulation in tomato fruits.previous studies of the genetic architecture of fruit metabolic composition have allowed us to identify four strongly conserved co-ordinate quantitative trait loci (qtl) for the branched-chain amino acids (bcaas). this study has been extended here to encompass the other 23 enzymes described to be involved in the pathways of bcaa synthesis and degradation. on coarse mapping the chromosomal location of these enzymes, it was possible to define the map position of 24 genes. of these genes eight co-l ...201121436187
bacterial transcription terminators: the rna 3'-end chronicles.the process of transcription termination is essential to proper expression of bacterial genes and, in many cases, to the regulation of bacterial gene expression. two types of bacterial transcriptional terminators are known to control gene expression. intrinsic terminators dissociate transcription complexes without the assistance of auxiliary factors. rho-dependent terminators are sites of dissociation mediated by an rna helicase called rho. despite decades of study, the molecular mechanisms of b ...201121439297
identification of local conformational similarity in structurally variable regions of homologous proteins using protein blocks.structure comparison tools can be used to align related protein structures to identify structurally conserved and variable regions and to infer functional and evolutionary relationships. while the conserved regions often superimpose well, the variable regions appear non superimposable. differences in homologous protein structures are thought to be due to evolutionary plasticity to accommodate diverged sequences during evolution. one of the kinds of differences between 3-d structures of homologou ...201121445259
evolution of introns in the archaeal world.the self-splicing group i introns are removed by an autocatalytic mechanism that involves a series of transesterification reactions. they require rna binding proteins to act as chaperones to correctly fold the rna into an active intermediate structure in vivo. pre-trna introns in bacteria and in higher eukaryote plastids are typical examples of self-splicing group i introns. by contrast, two striking features characterize rna splicing in the archaeal world. first, self-splicing group i introns c ...201121383132
how mutations in trna distant from the anticodon affect the fidelity of decoding.the ribosome converts genetic information into protein by selecting aminoacyl trnas whose anticodons base-pair to an mrna codon. mutations in the trna body can perturb this process and affect fidelity. the hirsh suppressor is a well-studied trna(trp) harboring a g24a mutation that allows readthrough of uga stop codons. here we present crystal structures of the 70s ribosome complexed with ef-tu and aminoacyl trna (native trna(trp), g24a trna(trp) or the miscoding a9c trna(trp)) bound to cognate u ...201121378964
the conformation of microrna seed regions in native micrornps is prearranged for presentation to mrna targets.micrornas control gene expression by post-transcriptional down-regulation of their target mrnas. complementarity between the seed region (nucleotides 2-8) of a microrna and the 3'-utr of its target mrna is the key determinant in recognition. however, the structural basis of the ability of the seed region to dominate target recognition in eukaryotic argonaute complexes has not been directly demonstrated. to better understand this problem, we performed chemical probing of micrornas held in native ...201121335607
comparative metagenomics of microbial traits within oceanic viral communities.viral genomes often contain genes recently acquired from microbes. in some cases (for example, psba) the proteins encoded by these genes have been shown to be important for viral replication. in this study, using a unique search strategy on the global ocean survey (gos) metagenomes in combination with marine virome and microbiome pyrosequencing-based datasets, we characterize previously undetected microbial metabolic capabilities concealed within the genomes of uncultured marine viral communitie ...201121307954
moderna: a tool for comparative modeling of rna 3d structure.rna is a large group of functionally important biomacromolecules. in striking analogy to proteins, the function of rna depends on its structure and dynamics, which in turn is encoded in the linear sequence. however, while there are numerous methods for computational prediction of protein three-dimensional (3d) structure from sequence, with comparative modeling being the most reliable approach, there are very few such methods for rna. here, we present moderna, a software tool for comparative mode ...201121300639
bidentate rna-magnesium clamps: on the origin of the special role of magnesium in rna folding.magnesium plays a special role in rna function and folding. although water is magnesium's most common first-shell ligand, the oxyanions of rna have significant affinity for magnesium. here we provide a quantum mechanical description of first-shell rna-magnesium and dna-magnesium interactions, demonstrating the unique features that characterize the energetics and geometry of magnesium complexes within large folded rnas. our work focuses on bidentate chelation of magnesium by rna or dna, where mul ...201121173199
interdependencies govern multidomain architecture in ribosomal small subunit assembly.the 30s subunit is composed of four structural domains, the body, platform, head, and penultimate/ultimate stems. the functional integrity of the 30s subunit is dependent upon appropriate assembly and precise orientation of all four domains. we examined 16s rrna conformational changes during in vitro assembly using directed hydroxyl radical probing mediated by fe(ii)-derivatized ribosomal protein (r-protein) s8. r-protein s8 binds the central domain of 16s rrna directly and independently and its ...201121156960
modeling rna polymerase competition: the effect of σ-subunit knockout and heat shock on gene transcription level.modeling of a complex biological process can explain the results of experimental studies and help predict its characteristics. among such processes is transcription in the presence of competing rna polymerases. this process involves rna polymerases collision followed by transcription termination.201121255416
crystal structures of complexes containing domains from two viral internal ribosome entry site (ires) rnas bound to the 70s ribosome.internal ribosome entry site (ires) rnas are elements of viral or cellular mrnas that bypass steps of canonical eukaryotic cap-dependent translation initiation. understanding of the structural basis of ires mechanisms is limited, partially due to a lack of high-resolution structures of ires rnas bound to their cellular targets. prompted by the universal phylogenetic conservation of the ribosomal p site, we solved the crystal structures of proposed p site binding domains from two intergenic regio ...201121245352
trna/mrna mimicry by tmrna and smpb in trans-translation.since accurate translation from mrna to protein is critical to survival, cells have developed translational quality control systems. bacterial ribosomes stalled on truncated mrna are rescued by a system involving tmrna and smpb referred to as trans-translation. here, we review current understanding of the mechanism of trans-translation. based on results obtained by using directed hydroxyl radical probing, we propose a new type of molecular mimicry during trans-translation. besides such chemical ...201121253384
crystallizing membrane proteins in lipidic mesophases. a host lipid screen.the default lipid for the bulk of the crystallogenesis studies performed to date using the cubic mesophase method is monoolein. there is no good reason however, why this 18-carbon, cis-monounsaturated monoacylglycerol should be the preferred lipid for all target membrane proteins. the latter come from an array of biomembrane types with varying properties that include hydrophobic thickness, intrinsic curvature, lateral pressure profile, lipid and protein makeup, and compositional asymmetry. thus, ...201121743796
dead-box proteins as rna helicases and chaperones.dead-box proteins are ubiquitous in rna-mediated processes and function by coupling cycles of atp binding and hydrolysis to changes in affinity for single-stranded rna. many dead-box proteins use this basic mechanism as the foundation for a version of rna helicase activity, efficiently separating the strands of short rna duplexes in a process that involves little or no translocation. this activity, coupled with mechanisms to direct different dead-box proteins to their physiological substrates, a ...201121297876
rna helicases at work: binding and rearranging.rna helicases are ubiquitous, highly conserved enzymes that participate in nearly all aspects of rna metabolism. these proteins bind or remodel rna or rna-protein complexes in an atp-dependent fashion. how rna helicases physically perform their cellular tasks has been a longstanding question, but in recent years, intriguing models have started to link structure, mechanism and biological function for some rna helicases. this review outlines our current view on major structural and mechanistic the ...201120813532
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
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