| viruses of haloarchaea. | in hypersaline environments, haloarchaea (halophilic members of the archaea) are the dominant organisms, and the viruses that infect them, haloarchaeoviruses are at least ten times more abundant. since their discovery in 1974, described haloarchaeoviruses include head-tailed, pleomorphic, spherical and spindle-shaped morphologies, representing myoviridae, siphoviridae, podoviridae, pleolipoviridae, sphaerolipoviridae and fuselloviridae families. this review overviews current knowledge of haloarc ... | 2014 | 25402735 |
| phylogenetically driven sequencing of extremely halophilic archaea reveals strategies for static and dynamic osmo-response. | organisms across the tree of life use a variety of mechanisms to respond to stress-inducing fluctuations in osmotic conditions. cellular response mechanisms and phenotypes associated with osmoadaptation also play important roles in bacterial virulence, human health, agricultural production and many other biological systems. to improve understanding of osmoadaptive strategies, we have generated 59 high-quality draft genomes for the haloarchaea (a euryarchaeal clade whose members thrive in hypersa ... | 2014 | 25393412 |
| structural insight into amino group-carrier protein-mediated lysine biosynthesis: crystal structure of the lysz·lysw complex from thermus thermophilus. | in the biosynthesis of lysine by thermus thermophilus, the metabolite α-ketoglutarate is converted to the intermediate α-aminoadipate (aaa), which is protected by the 54-amino acid acidic protein lysw. in this study, we determined the crystal structure of lysz from t. thermophilus (ttlysz), an amino acid kinase that catalyzes the second step in the aaa to lysine conversion, which was in a complex with lysw at a resolution of 1.85 å. a crystal analysis coupled with isothermal titration calorimetr ... | 2014 | 25392000 |
| structural insight into amino group-carrier protein-mediated lysine biosynthesis: crystal structure of the lysz·lysw complex from thermus thermophilus. | in the biosynthesis of lysine by thermus thermophilus, the metabolite α-ketoglutarate is converted to the intermediate α-aminoadipate (aaa), which is protected by the 54-amino acid acidic protein lysw. in this study, we determined the crystal structure of lysz from t. thermophilus (ttlysz), an amino acid kinase that catalyzes the second step in the aaa to lysine conversion, which was in a complex with lysw at a resolution of 1.85 å. a crystal analysis coupled with isothermal titration calorimetr ... | 2014 | 25392000 |
| cecafdb: a curated database for the documentation, visualization and comparative analysis of central carbon metabolic flux distributions explored by 13c-fluxomics. | the central carbon metabolic flux database (cecafdb, available at http://www.cecafdb.org) is a manually curated, multipurpose and open-access database for the documentation, visualization and comparative analysis of the quantitative flux results of central carbon metabolism among microbes and animal cells. it encompasses records for more than 500 flux distributions among 36 organisms and includes information regarding the genotype, culture medium, growth conditions and other specific information ... | 2014 | 25392417 |
| cecafdb: a curated database for the documentation, visualization and comparative analysis of central carbon metabolic flux distributions explored by 13c-fluxomics. | the central carbon metabolic flux database (cecafdb, available at http://www.cecafdb.org) is a manually curated, multipurpose and open-access database for the documentation, visualization and comparative analysis of the quantitative flux results of central carbon metabolism among microbes and animal cells. it encompasses records for more than 500 flux distributions among 36 organisms and includes information regarding the genotype, culture medium, growth conditions and other specific information ... | 2014 | 25392417 |
| thermodynamic system drift in protein evolution. | proteins from thermophiles are generally more thermostable than their mesophilic homologs, but little is known about the evolutionary process driving these differences. here we attempt to understand how the diverse thermostabilities of bacterial ribonuclease h1 (rnh) proteins evolved. rnh proteins from thermus thermophilus (ttrnh) and escherichia coli (ecrnh) share similar structures but differ in melting temperature (t(m)) by 20 °c. ttrnh's greater stability is caused in part by the presence of ... | 2014 | 25386647 |
| interacting cytoplasmic loops of subunits a and c of escherichia coli f1f0 atp synthase gate h+ transport to the cytoplasm. | h(+)-transporting f1f0 atp synthase catalyzes the synthesis of atp via coupled rotary motors within f0 and f1. h(+) transport at the subunit a-c interface in transmembranous f0 drives rotation of a cylindrical c10 oligomer within the membrane, which is coupled to rotation of subunit γ within the α3β3 sector of f1 to mechanically drive atp synthesis. f1f0 functions in a reversible manner, with atp hydrolysis driving h(+) transport. atp-driven h(+) transport in a select group of cysteine mutants i ... | 2014 | 25385585 |
| phenotyping the quality of complex medium components by simple online-monitored shake flask experiments. | media containing yeast extracts and other complex raw materials are widely used for the cultivation of microorganisms. however, variations in the specific nutrient composition can occur, due to differences in the complex raw material ingredients and in the production of these components. these lot-to-lot variations can affect growth rate, product yield and product quality in laboratory investigations and biopharmaceutical production processes. in the fda's process analytical technology (pat) ini ... | 2014 | 25376163 |
| the σ enigma: bacterial σ factors, archaeal tfb and eukaryotic tfiib are homologs. | structural comparisons of initiating rna polymerase complexes and structure-based amino acid sequence alignments of general transcription initiation factors (eukaryotic tfiib, archaeal tfb and bacterial σ factors) show that these proteins are homologs. tfiib and tfb each have two-five-helix cyclin-like repeats (clrs) that include a c-terminal helix-turn-helix (hth) motif (clr/hth domains). four homologous hth motifs are present in bacterial σ factors that are relics of clr/hth domains. sequence ... | 2014 | 25483602 |
| rna targeting by the type iii-a crispr-cas csm complex of thermus thermophilus. | crispr-cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. here we report the structure and function of the effector complex of the type iii-a crispr-cas system of thermus thermophilus: the csm complex (ttcsm). ttcsm is composed of five different protein subunits (csm1-csm5) with an uneven stoichiometry and a single crrna of variable size (35-53 nt). the ttcsm crrna content is similar to the type iii-b cmr complex, indicating that cr ... | 2014 | 25457165 |
| silencing by h-ns potentiated the evolution of salmonella. | the bacterial h-ns protein silences expression from sequences with higher at-content than the host genome and is believed to buffer the fitness consequences associated with foreign gene acquisition. loss of h-ns results in severe growth defects in salmonella, but the underlying reasons were unclear. an experimental evolution approach was employed to determine which secondary mutations could compensate for the loss of h-ns in salmonella. six independently derived s. typhimurium hns mutant strains ... | 2014 | 25375226 |
| multilayered polyelectrolyte microcapsules: interaction with the enzyme cytochrome c oxidase. | cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. the interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. we found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsu ... | 2014 | 25372607 |
| diversity and regulation of atp sulfurylase in photosynthetic organisms. | atp sulfurylase (atps) catalyzes the first committed step in the sulfate assimilation pathway, the activation of sulfate prior to its reduction. atps has been studied in only a few model organisms and even in these cases to a much smaller extent than the sulfate reduction and cysteine synthesis enzymes. this is possibly because the latter were considered of greater regulatory importance for sulfate assimilation. recent evidences (reported in this paper) challenge this view and suggest that atps ... | 2014 | 25414712 |
| studying the active-site loop movement of the são paolo metallo-β-lactamase-1†electronic supplementary information (esi) available: procedures for protein expression and purification, (19)f-labelling, crystallisation, data collection, and structure determination, table of crystallographic data, table of crystallographic parameters and refinement statistics, figures showing binding mode and distances, procedures for mass spectrometry measurements, differential scanning fluorimetry measurements, stopped-flow measurements and other kinetics measurements. see doi: 10.1039/c4sc01752hclick here for additional data file. | metallo-β-lactamases (mbls) catalyse the hydrolysis of almost all β-lactam antibiotics. we report biophysical and kinetic studies on the são paulo mbl (spm-1), which reveal its zn(ii) ion usage and mechanism as characteristic of the clinically important di-zn(ii) dependent b1 mbl subfamily. biophysical analyses employing crystallography, dynamic (19)f nmr and ion mobility mass spectrometry, however, reveal that spm-1 possesses loop and mobile element regions characteristic of the b2 mbls. these ... | 2014 | 25717359 |
| studying the active-site loop movement of the são paolo metallo-β-lactamase-1†electronic supplementary information (esi) available: procedures for protein expression and purification, (19)f-labelling, crystallisation, data collection, and structure determination, table of crystallographic data, table of crystallographic parameters and refinement statistics, figures showing binding mode and distances, procedures for mass spectrometry measurements, differential scanning fluorimetry measurements, stopped-flow measurements and other kinetics measurements. see doi: 10.1039/c4sc01752hclick here for additional data file. | metallo-β-lactamases (mbls) catalyse the hydrolysis of almost all β-lactam antibiotics. we report biophysical and kinetic studies on the são paulo mbl (spm-1), which reveal its zn(ii) ion usage and mechanism as characteristic of the clinically important di-zn(ii) dependent b1 mbl subfamily. biophysical analyses employing crystallography, dynamic (19)f nmr and ion mobility mass spectrometry, however, reveal that spm-1 possesses loop and mobile element regions characteristic of the b2 mbls. these ... | 2014 | 25717359 |
| structural basis for ion selectivity revealed by high-resolution crystal structure of mg2+ channel mgte. | magnesium is the most abundant divalent cation in living cells and is crucial to several biological processes. mgte is a mg(2+) channel distributed in all domains of life that contributes to the maintenance of cellular mg(2+) homeostasis. here we report the high-resolution crystal structures of the transmembrane domain of mgte, bound to mg(2+), mn(2+) and ca(2+). the high-resolution mg(2+)-bound crystal structure clearly visualized the hydrated mg(2+) ion within its selectivity filter. based on ... | 2014 | 25367295 |
| proton transfer in the k-channel analog of b-type cytochrome c oxidase from thermus thermophilus. | a key enzyme in aerobic metabolism is cytochrome c oxidase (cco), which catalyzes the reduction of molecular oxygen to water in the mitochondrial and bacterial membranes. substrate electrons and protons are taken up from different sides of the membrane and protons are pumped across the membrane, thereby generating an electrochemical gradient. the well-studied a-type cco uses two different entry channels for protons: the d-channel for all pumped and two consumed protons, and the k-channel for the ... | 2014 | 25418102 |
| molecular insights into dna interference by crispr-associated nuclease-helicase cas3. | mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (crisprs) and crispr-associated (cas) proteins. type i crispr-cas systems use a "cascade" ribonucleoprotein complex to guide rna specifically to complementary sequence in invader double-stranded dna (dsdna), a process called "interference." after target recognition by cascade, formation of an r-loop triggers recruitment of a cas3 nuclease-helicase, completing the int ... | 2014 | 25368186 |
| subnanometre-resolution electron cryomicroscopy structure of a heterodimeric abc exporter. | atp-binding cassette (abc) transporters translocate substrates across cell membranes, using energy harnessed from atp binding and hydrolysis at their nucleotide-binding domains. abc exporters are present both in prokaryotes and eukaryotes, with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases. tmrab is a heterodimeric abc exporter from the thermophilic gram-negative eubacterium thermus thermophilus; it is homologous to various multidrug ... | 2014 | 25363761 |
| subnanometre-resolution electron cryomicroscopy structure of a heterodimeric abc exporter. | atp-binding cassette (abc) transporters translocate substrates across cell membranes, using energy harnessed from atp binding and hydrolysis at their nucleotide-binding domains. abc exporters are present both in prokaryotes and eukaryotes, with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases. tmrab is a heterodimeric abc exporter from the thermophilic gram-negative eubacterium thermus thermophilus; it is homologous to various multidrug ... | 2014 | 25363761 |
| defect in the formation of 70s ribosomes caused by lack of ribosomal protein l34 can be suppressed by magnesium. | to elucidate the biological functions of the ribosomal protein l34, which is encoded by the rpmh gene, the rpmh deletion mutant of bacillus subtilis and two suppressor mutants were characterized. although the δrpmh mutant exhibited a severe slow-growth phenotype, additional mutations in the yhdp or mgte gene restored the growth rate of the δrpmh strain. either the disruption of yhdp, which is thought to be involved in the efflux of mg(2+), or overexpression of mgte, which plays a major role in t ... | 2014 | 25182490 |
| phenotypic interactions among mutations in a thermus thermophilus 16s rrna gene detected with genetic selections and experimental evolution. | during protein synthesis, the ribosome undergoes conformational transitions between functional states, requiring communication between distant structural elements of the ribosome. despite advances in ribosome structural biology, identifying the protein and rrna residues governing these transitions remains a significant challenge. such residues can potentially be identified genetically, given the predicted deleterious effects of mutations stabilizing the ribosome in discrete conformations and the ... | 2014 | 25157075 |
| dissection of the region of pseudomonas aeruginosa para that is important for dimerization and interactions with its partner parb. | pseudomonas aeruginosa para belongs to a large subfamily of walker-type atpases acting as partitioning proteins in bacteria. para has the ability to both self-associate and interact with its partner parb. analysis of the deletion mutants defined the part of the protein involved in dimerization and interactions with parb. here, a set of para alanine substitution mutants in the region between e67 and l85 was created and analysed in vivo and in vitro. all mutants impaired in dimerization (substitut ... | 2014 | 25139949 |
| target rna capture and cleavage by the cmr type iii-b crispr-cas effector complex. | the effector complex of the cmr/type iii-b crispr (clustered regularly interspaced short palindromic repeat)-cas (crispr-associated) system cleaves rnas recognized by the crispr rna (crrna) of the complex and includes six protein subunits of unknown functions. using reconstituted pyrococcus furiosus cmr complexes, we found that each of the six cmr proteins plays a critical role in either crrna interaction or target rna capture. cmr2, cmr3, cmr4, and cmr5 are all required for formation of a crrna ... | 2014 | 25367038 |
| rejection of tmrna·smpb after gtp hydrolysis by ef-tu on ribosomes stalled on intact mrna. | messenger rnas lacking a stop codon trap ribosomes at their 3' ends, depleting the pool of ribosomes available for protein synthesis. in bacteria, a remarkable quality control system rescues and recycles stalled ribosomes in a process known as trans-translation. acting as a trna, transfer-messenger rna (tmrna) is aminoacylated, delivered by ef-tu to the ribosomal a site, and accepts the nascent polypeptide. translation then resumes on a reading frame within tmrna, encoding a short peptide tag th ... | 2014 | 25246654 |
| structure and mechanism of e. coli rna 2',3'-cyclic phosphodiesterase. | 2h (two-histidine) phosphoesterase enzymes are distributed widely in all domains of life and are implicated in diverse rna and nucleotide transactions, including the transesterification and hydrolysis of cyclic phosphates. here we report a biochemical and structural characterization of the escherichia coli 2h protein yapd yadp [corrected], which was identified originally as a reversible transesterifying "nuclease/ligase" at rna 2',5'-phosphodiesters. we find that yapd yadp [corrected] is an "end ... | 2014 | 25239919 |
| rna. complete pairing not needed. | | 2014 | 25359948 |
| interplay between e. coli dnak, clpb and grpe during protein disaggregation. | the dnak/hsp70 chaperone system and clpb/hsp104 collaboratively disaggregate protein aggregates and reactivate inactive proteins. the teamwork is specific: escherichia coli dnak interacts with e. coli clpb and yeast hsp70, ssa1, interacts with yeast hsp104. this interaction is between the middle domains of hexameric clpb/hsp104 and the dnak/hsp70 nucleotide-binding domain (nbd). to identify the site on e. coli dnak that interacts with clpb, we substituted amino acid residues throughout the dnak ... | 2014 | 25451597 |
| interplay between e. coli dnak, clpb and grpe during protein disaggregation. | the dnak/hsp70 chaperone system and clpb/hsp104 collaboratively disaggregate protein aggregates and reactivate inactive proteins. the teamwork is specific: escherichia coli dnak interacts with e. coli clpb and yeast hsp70, ssa1, interacts with yeast hsp104. this interaction is between the middle domains of hexameric clpb/hsp104 and the dnak/hsp70 nucleotide-binding domain (nbd). to identify the site on e. coli dnak that interacts with clpb, we substituted amino acid residues throughout the dnak ... | 2014 | 25451597 |
| arfa recognizes the lack of mrna in the mrna channel after rf2 binding for ribosome rescue. | although trans-translation mediated by tmrna-smpb has long been known as the sole system to relieve bacterial stalled ribosomes, arfa has recently been identified as an alternative factor for ribosome rescue in escherichia coli. this process requires hydrolysis of nascent peptidyl-trna by rf2, which usually acts as a stop codon-specific peptide release factor. it poses a fascinating question of how arfa and rf2 recognize and rescue the stalled ribosome. here, we mapped the location of arfa in th ... | 2014 | 25355516 |
| the mechanism of torsin atpase activation. | torsins are membrane-associated atpases whose activity is dependent on two activating cofactors, lamina-associated polypeptide 1 (lap1) and luminal domain-like lap1 (lull1). the mechanism by which these cofactors regulate torsin activity has so far remained elusive. in this study, we identify a conserved domain in these activators that is predicted to adopt a fold resembling an aaa+ (atpase associated with a variety of cellular activities) domain. within these domains, a strictly conserved arg r ... | 2014 | 25352667 |
| structure of type ii dehydroquinase from pseudomonas aeruginosa. | pseudomonas aeruginosa causes opportunistic infections and is resistant to most antibiotics. ongoing efforts to generate much-needed new antibiotics include targeting enzymes that are vital for p. aeruginosa but are absent in mammals. one such enzyme, type ii dehydroquinase (dhqase), catalyzes the interconversion of 3-dehydroquinate and 3-dehydroshikimate, a necessary step in the shikimate pathway. this step is vital for the proper synthesis of phenylalanine, tryptophan, tyrosine and other aroma ... | 2014 | 25372814 |
| temperature-dependent radiation sensitivity and order of 70s ribosome crystals. | all evidence to date indicates that at t = 100 k all protein crystals exhibit comparable sensitivity to x-ray damage when quantified using global metrics such as change in scaling b factor or integrated intensity versus dose. this is consistent with observations in cryo-electron microscopy, and results because nearly all diffusive motions of protein and solvent, including motions induced by radiation damage, are frozen out. but how do the sensitivities of different proteins compare at room tempe ... | 2014 | 25372680 |
| trigger-helix folding pathway and si3 mediate catalysis and hairpin-stabilized pausing by escherichia coli rna polymerase. | the conformational dynamics of the polymorphous trigger loop (tl) in rna polymerase (rnap) underlie multiple steps in the nucleotide addition cycle and diverse regulatory mechanisms. these mechanisms include nascent rna hairpin-stabilized pausing, which inhibits tl folding into the trigger helices (th) required for rapid nucleotide addition. the nascent rna pause hairpin forms in the rna exit channel and promotes opening of the rnap clamp domain, which in turn stabilizes a partially folded, paus ... | 2014 | 25336618 |
| noncanonical cell-to-cell dna transfer in thermus spp. is insensitive to argonaute-mediated interference. | horizontal gene transfer drives the rapid evolution of bacterial populations. classical processes that promote the lateral flow of genetic information are conserved throughout the prokaryotic world. however, some species have nonconserved transfer mechanisms that are not well known. this is the case for the ancient extreme thermophile thermus thermophilus. in this work, we show that t. thermophilus strains are capable of exchanging large dna fragments by a novel mechanism that requires cell-to-c ... | 2014 | 25331432 |
| noncanonical cell-to-cell dna transfer in thermus spp. is insensitive to argonaute-mediated interference. | horizontal gene transfer drives the rapid evolution of bacterial populations. classical processes that promote the lateral flow of genetic information are conserved throughout the prokaryotic world. however, some species have nonconserved transfer mechanisms that are not well known. this is the case for the ancient extreme thermophile thermus thermophilus. in this work, we show that t. thermophilus strains are capable of exchanging large dna fragments by a novel mechanism that requires cell-to-c ... | 2014 | 25331432 |
| rna-protein distance patterns in ribosomes reveal the mechanism of translational attenuation. | elucidating protein translational regulation is crucial for understanding cellular function and drug development. a key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. the structure and dynamics of ribosome complexes were resolved recently thanks to the development of x-ray crystallography, cryo-em, and single molecule biophysics. current studies of the ribosome have shown multiple functional states, each with a un ... | 2014 | 25326828 |
| molecular dynamics simulations of the nip7 proteins from the marine deep- and shallow-water pyrococcus species. | the identification of the mechanisms of adaptation of protein structures to extreme environmental conditions is a challenging task of structural biology. we performed molecular dynamics (md) simulations of the nip7 protein involved in rna processing from the shallow-water (p. furiosus) and the deep-water (p. abyssi) marine hyperthermophylic archaea at different temperatures (300 and 373 k) and pressures (0.1, 50 and 100 mpa). the aim was to disclose similarities and differences between the deep- ... | 2014 | 25315147 |
| translation initiation factor 3 regulates switching between different modes of ribosomal subunit joining. | ribosomal subunit joining is a key checkpoint in the bacterial translation initiation pathway during which initiation factors (ifs) regulate association of the 30s initiation complex (ic) with the 50s subunit to control formation of a 70s ic that can enter into the elongation stage of protein synthesis. the gtp-bound form of if2 accelerates subunit joining, whereas if3 antagonizes subunit joining and plays a prominent role in maintaining translation initiation fidelity. the molecular mechanisms ... | 2014 | 25308340 |
| translation initiation factor 3 regulates switching between different modes of ribosomal subunit joining. | ribosomal subunit joining is a key checkpoint in the bacterial translation initiation pathway during which initiation factors (ifs) regulate association of the 30s initiation complex (ic) with the 50s subunit to control formation of a 70s ic that can enter into the elongation stage of protein synthesis. the gtp-bound form of if2 accelerates subunit joining, whereas if3 antagonizes subunit joining and plays a prominent role in maintaining translation initiation fidelity. the molecular mechanisms ... | 2014 | 25308340 |
| structure and mechanism of the bifunctional cina enzyme from thermus thermophilus. | cina is a widely distributed protein in gram-positive and gram-negative bacteria. it is associated with natural competence and is proposed to have a function as an enzyme participating in the pyridine nucleotide cycle, which recycles products formed by non-redox uses of nad. here we report the determination of the crystal structure of cina from thermus thermophilus, in complex with several ligands. cina was shown to have both nicotinamide mononucleotide deamidase and adp-ribose pyrophosphatase a ... | 2014 | 25313401 |
| chemical probing of rna with the hydroxyl radical at single-atom resolution. | while hydroxyl radical cleavage is widely used to map rna tertiary structure, lack of mechanistic understanding of strand break formation limits the degree of structural insight that can be obtained from this experiment. here, we determine how individual ribose hydrogens of sarcin/ricin loop rna participate in strand cleavage. we find that substituting deuterium for hydrogen at a ribose 5'-carbon produces a kinetic isotope effect on cleavage; the major cleavage product is an rna strand terminate ... | 2014 | 25313156 |
| structural characterization of the putative abc-type 2 transporter from thermotoga maritima msb8. | this study describes the structure of the putative abc-type 2 transporter tm0543 from thermotoga maritima msb8 determined at a resolution of 2.3 å. in comparative sequence-clustering analysis, tm0543 displays similarity to natab-like proteins, which are components of the abc-type na(+) efflux pump permease. however, the overall structure fold of the predicted nucleotide-binding domain reveals that it is different from any known structure of abc-type efflux transporters solved to date. the struct ... | 2014 | 25306867 |
| negamycin interferes with decoding and translocation by simultaneous interaction with rrna and trna. | negamycin (neg) is a ribosome-targeting antibiotic that exhibits clinically promising activity. its binding site and mode of action have remained unknown. we solved the structure of the thermus thermophilus ribosome bound to mrna and three trnas, in complex with neg. the drug binds to both small and large ribosomal subunits at nine independent sites. resistance mutations in the 16s rrna unequivocally identified the binding site in the vicinity of the conserved helix 34 (h34) in the small subunit ... | 2014 | 25306922 |
| amicoumacin a inhibits translation by stabilizing mrna interaction with the ribosome. | we demonstrate that the antibiotic amicoumacin a (ami) is a potent inhibitor of protein synthesis. resistance mutations in helix 24 of the 16s rrna mapped the ami binding site to the small ribosomal subunit. the crystal structure of bacterial ribosome in complex with ami solved at 2.4 å resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16s rrna in the e site and the mrna backbone. simultaneous interactions of ami with 16s rrna and mrna and the in vi ... | 2014 | 25306919 |
| versatility of acyl-acyl carrier protein synthetases. | the acyl carrier protein (acp) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in acp-dependent metabolic pathways. we show that acyl-acp synthetases (aasss) from different organisms are able to load even, odd, and unnatural fatty acids onto e. coli acp in vitro. vibrio harveyi aass not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier protein ... | 2014 | 25308274 |
| structure of the large ribosomal subunit from human mitochondria. | human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. they are linked with hereditary mitochondrial diseases and are often the unintended targets of various clinically useful antibiotics. using single-particle cryogenic electron microscopy, we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. the structure unveil ... | 2014 | 25278503 |
| the k-turn motif in riboswitches and other rna species. | the kink turn is a widespread structure motif that introduces a tight bend into the axis of duplex rna. this generally functions to mediate tertiary interactions, and to serve as a specific protein binding site. k-turns or closely related structures are found in at least seven different riboswitch structures, where they function as key architectural elements that help generate the ligand binding pocket. this article is part of a special issue entitled: riboswitches. | 2014 | 24798078 |
| overview of cell-free protein synthesis: historic landmarks, commercial systems, and expanding applications. | during the early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger rna. owing to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. commercial cell-free systems are now available from a variety of material sources, ranging from "traditional" e. coli, rabbit ... | 2014 | 25271714 |
| in vivo tmrna protection by smpb and pre-ribosome binding conformation in solution. | tmrna is an abundant rna in bacteria with trna and mrna features. it is specialized in trans-translation, a translation rescuing system. we demonstrate that its partner protein smpb binds the trna-like region (tld) in vivo and chaperones the fold of the tld-h2 region. we use an original approach combining the observation of tmrna degradation pathways in a heterologous system, the analysis of the tmrna digests by ms and nmr, and co-overproduction assays of tmrna and smpb. we study the conformatio ... | 2014 | 25135523 |
| the bioconversion of red ginseng ethanol extract into compound k by saccharomyces cerevisiae hj-014. | a β-glucosidase producing yeast strain was isolated from korean traditional rice wine. based on the sequence of the ycl008c gene and analysis of the fatty acid composition, the isolate was identified as saccharomyces cerevisiae strain hj-014. s. cerevisiae hj-014 produced ginsenoside rd, f2, and compound k from the ethanol extract of red ginseng. the production was increased by shaking culture, where the bioconversion efficiency was increased 2-fold compared to standing culture. the production o ... | 2014 | 25346602 |
| identification and evaluation of improved 4'-o-(alkyl) 4,5-disubstituted 2-deoxystreptamines as next-generation aminoglycoside antibiotics. | the emerging epidemic of drug resistance places the development of efficacious and safe antibiotics in the spotlight of current research. here, we report the design of next-generation aminoglycosides. discovery efforts were driven by rational synthesis focusing on 4' alkylations of the aminoglycoside paromomycin, with the goal to alleviate the most severe and disabling side effect of aminoglycosides-irreversible hearing loss. compounds were evaluated for target activity in in vitro ribosomal tra ... | 2014 | 25271289 |
| speciation and reactivity of uranium products formed during in situ bioremediation in a shallow alluvial aquifer. | in this study, we report the results of in situ u(vi) bioreduction experiments at the integrated field research challenge site in rifle, colorado, usa. columns filled with sediments were deployed into a groundwater well at the site and, after a period of conditioning with groundwater, were amended with a mixture of groundwater, soluble u(vi), and acetate to stimulate the growth of indigenous microorganisms. individual reactors were collected as various redox regimes in the column sediments were ... | 2014 | 25265543 |
| sequence co-evolution gives 3d contacts and structures of protein complexes. | protein-protein interactions are fundamental to many biological processes. experimental screens have identified tens of thousands of interactions, and structural biology has provided detailed functional insight for select 3d protein complexes. an alternative rich source of information about protein interactions is the evolutionary sequence record. building on earlier work, we show that analysis of correlated evolutionary sequence changes across proteins identifies residues that are close in spac ... | 2014 | 25255213 |
| torque generation of enterococcus hirae v-atpase. | v-atpase (v(o)v1) converts the chemical free energy of atp into an ion-motive force across the cell membrane via mechanical rotation. this energy conversion requires proper interactions between the rotor and stator in v(o)v1 for tight coupling among chemical reaction, torque generation, and ion transport. we developed an escherichia coli expression system for enterococcus hirae v(o)v1 (ehv(o)v1) and established a single-molecule rotation assay to measure the torque generated. recombinant and nat ... | 2014 | 25258315 |
| evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule. | the molten globule, a conformational ensemble with significant secondary structure but only loosely packed tertiary structure, has been suggested to be a ubiquitous intermediate in protein folding. however, it is difficult to assess the tertiary packing of transiently populated species to evaluate this hypothesis. escherichia coli rnase h is known to populate an intermediate before the rate-limiting barrier to folding that has long been thought to be a molten globule. we investigated this hypoth ... | 2014 | 25258414 |
| analysis of genomic rearrangements, horizontal gene transfer and role of plasmids in the evolution of industrial important thermus species. | bacteria of genus thermus inhabit both man-made and natural thermal environments. several thermus species have shown biotechnological potential such as reduction of heavy metals which is essential for eradication of heavy metal pollution; removing of organic contaminants in water; opening clogged pipes, controlling global warming among many others. enzymes from thermophilic bacteria have exhibited higher activity and stability than synthetic or enzymes from mesophilic organisms. | 2014 | 25257245 |
| inactivation and unfolding of protein tyrosine phosphatase from thermus thermophilus hb27 during urea and guanidine hydrochloride denaturation. | the effects of urea and guanidine hydrochloride (gdnhcl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (ptpase), a thermostable low molecular weight protein from thermus thermophilus hb27, have been studied. enzymatic activity assays showed both urea and gdnhcl resulted in the inactivation of ptpase in a concentration and time-dependent manner. inactivation kinetics analysis suggested that the inactivation of ptpase induced by urea and gdnhcl were both monop ... | 2014 | 25255086 |
| trans-acting arginine residues in the aaa+ chaperone clpb allosterically regulate the activity through inter- and intradomain communication. | the molecular chaperone clpb/hsp104, a member of the aaa+ superfamily (atpases associated with various cellular activities), rescues proteins from the aggregated state in collaboration with the dnak/hsp70 chaperone system. clpb/hsp104 forms a hexameric, ring-shaped complex that functions as a tightly regulated, atp-powered molecular disaggregation machine. highly conserved and essential arginine residues, often called arginine fingers, are located at the subunit interfaces of the complex, which ... | 2014 | 25253689 |
| ribosome-induced tuning of gtp hydrolysis by a translational gtpase. | gtp hydrolysis by elongation factor tu (ef-tu), a translational gtpase that delivers aminoacyl-trnas to the ribosome, plays a crucial role in decoding and translational fidelity. the basic reaction mechanism and the way the ribosome contributes to catalysis are a matter of debate. here we use mutational analysis in combination with measurements of rate/ph profiles, kinetic solvent isotope effects, and ion dependence of gtp hydrolysis by ef-tu off and on the ribosome to dissect the reaction mecha ... | 2014 | 25246550 |
| severe mgadp inhibition of bacillus subtilis f1-atpase is not due to the absence of nucleotide binding to the noncatalytic nucleotide binding sites. | f1-atpase from bacillus subtilis (bf1) is severely suppressed by the mgadp inhibition. here, we have tested if this is due to the loss of nucleotide binding to the noncatalytic site that is required for the activation. measurements with a tryptophan mutant of bf1 indicated that the noncatalytic sites could bind atp normally. furthermore, the mutant bf1 that cannot bind atp to the noncatalytic sites showed much lower atpase activity. it was concluded that the cause of strong mgadp inhibition of b ... | 2014 | 25244289 |
| iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery. | iron-sulfur (fe-s) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. the combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables fe-s clusters to participate in numerous biological processes. fe-s clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i.e. ... | 2014 | 25245479 |
| iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery. | iron-sulfur (fe-s) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. the combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables fe-s clusters to participate in numerous biological processes. fe-s clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i.e. ... | 2014 | 25245479 |
| ribosome rescue and translation termination at non-standard stop codons by ict1 in mammalian mitochondria. | release factors (rfs) govern the termination phase of protein synthesis. human mitochondria harbor four different members of the class 1 rf family: rf1lmt/mtrf1a, rf1mt, c12orf65 and ict1. the homolog of the essential ict1 factor is widely distributed in bacteria and organelles and has the peculiar feature in human mitochondria to be part of the ribosome as a ribosomal protein of the large subunit. the factor has been suggested to rescue stalled ribosomes in a codon-independent manner. the mecha ... | 2014 | 25233460 |
| dynamics of rna modification by a multi-site-specific trna methyltransferase. | in most organisms, the widely conserved 1-methyl-adenosine58 (m1a58) trna modification is catalyzed by an s-adenosyl-l-methionine (sam)-dependent, site-specific enzyme trmi. in archaea, trmi also methylates the adjacent adenine 57, m1a57 being an obligatory intermediate of 1-methyl-inosine57 formation. to study this multi-site specificity, we used three oligoribonucleotide substrates of pyrococcus abyssi trmi (pabtrmi) containing a fluorescent 2-aminopurine (2-ap) at the two target positions and ... | 2014 | 25217588 |
| macrolide-peptide conjugates as probes of the path of travel of the nascent peptides through the ribosome. | despite decades of research on the bacterial ribosome, the ribosomal exit tunnel is still poorly understood. although it has been suggested that the exit tunnel is simply a convenient route of egress for the nascent chain, specific protein sequences serve to slow the rate of translation, suggesting some degree of interaction between the nascent peptide chain and the exit tunnel. to understand how the ribosome interacts with nascent peptide sequences, we synthesized and characterized a novel clas ... | 2014 | 25198768 |
| architecture of mammalian respiratory complex i. | complex i (nadh:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. it couples electron transfer from nadh to ubiquinone with proton translocation across the energy-transducing inner membrane, providing electrons for respiration and driving atp synthesis. mammalian complex i contains 44 different nuclear- and mitochondrial-encoded subunits, with a combined mass of 1 mda. the 14 conserved 'core' subunits have been structurally defined in the minimal, b ... | 2014 | 25209663 |
| the evolution of respiratory o2/no reductases: an out-of-the-phylogenetic-box perspective. | complex life on our planet crucially depends on strong redox disequilibria afforded by the almost ubiquitous presence of highly oxidizing molecular oxygen. however, the history of o2-levels in the atmosphere is complex and prior to the great oxidation event some 2.3 billion years ago, the amount of o2 in the biosphere is considered to have been extremely low as compared with present-day values. therefore the evolutionary histories of life and of o2-levels are likely intricately intertwined. the ... | 2014 | 24968694 |
| molecular mechanics of 30s subunit head rotation. | during ribosomal translocation, a process central to the elongation phase of protein synthesis, movement of mrna and trnas requires large-scale rotation of the head domain of the small (30s) subunit of the ribosome. it has generally been accepted that the head rotates by pivoting around the neck helix (h28) of 16s rrna, its sole covalent connection to the body domain. surprisingly, we observe that the calculated axis of rotation does not coincide with the neck. instead, comparative structure ana ... | 2014 | 25187561 |
| bacterial sigma factors as targets for engineered or synthetic transcriptional control. | sigma (σ) factors are the predominant constituents of transcription regulation in bacteria. σ factors recruit the core rna polymerase to recognize promoters with specific dna sequences. recently, engineering of transcriptional regulators has become a significant tool for strain engineering. the present review summarizes the recent advances in σ factor based engineering or synthetic design. the manipulation of σ factors presents insights into the bacterial stress tolerance and metabolite producti ... | 2014 | 25232540 |
| the smpb c-terminal tail helps tmrna to recognize and enter stalled ribosomes. | in bacteria, transfer-messenger rna (tmrna) and smpb comprise the most common and effective system for rescuing stalled ribosomes. ribosomes stall on mrna transcripts lacking stop codons and are rescued as the defective mrna is swapped for the tmrna template in a process known as trans-translation. the tmrna-smpb complex is recruited to the ribosome independent of a codon-anticodon interaction. given that the ribosome uses robust discriminatory mechanisms to select against non-cognate trnas duri ... | 2014 | 25228900 |
| the evolutionary journey of argonaute proteins. | argonaute proteins are conserved throughout all domains of life. recently characterized prokaryotic argonaute proteins (pagos) participate in host defense by dna interference, whereas eukaryotic argonaute proteins (eagos) control a wide range of processes by rna interference. here we review molecular mechanisms of guide and target binding by argonaute proteins, and describe how the conformational changes induced by target binding lead to target cleavage. on the basis of structural comparisons an ... | 2014 | 25192263 |
| heteronuclear transverse and longitudinal relaxation in ax4 spin systems: application to (15)n relaxations in (15)nh4(+). | the equations that describe the time-evolution of transverse and longitudinal (15)n magnetisations in tetrahedral ammonium ions, (15)nh4(+), are derived from the bloch-wangsness-redfield density operator relaxation theory. it is assumed that the relaxation of the spin-states is dominated by (1) the intra-molecular (15)n-(1)h and (1)h-(1)h dipole-dipole interactions and (2) interactions of the ammonium protons with remote spins, which also include the contribution to the relaxations that arise fr ... | 2014 | 25128779 |
| acetylome analysis reveals diverse functions of lysine acetylation in mycobacterium tuberculosis. | the lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of m. tuberculosis. however, only a few acetylated proteins of m. tuberculosis are known, presenting a major obstacle to understanding the ... | 2014 | 25180227 |
| holliday junction resolvases. | four-way dna intermediates, called holliday junctions (hjs), can form during meiotic and mitotic recombination, and their removal is crucial for chromosome segregation. a group of ubiquitous and highly specialized structure-selective endonucleases catalyze the cleavage of hjs into two disconnected dna duplexes in a reaction called hj resolution. these enzymes, called hj resolvases, have been identified in bacteria and their bacteriophages, archaea, and eukaryotes. in this review, we discuss fund ... | 2014 | 25183833 |
| a bridge between the aminoacylation and editing domains of leucyl-trna synthetase is crucial for its synthetic activity. | leucyl-trna synthetases (leurss) catalyze the linkage of leucine with trna(leu). leurs contains a catalysis domain (aminoacylation) and a cp1 domain (editing). cp1 is inserted 35 å from the aminoacylation domain. aminoacylation and editing require cp1 to swing to the coordinated conformation. the neck between the cp1 domain and the aminoacylation domain is defined as the cp1 hairpin. the location of the cp1 hairpin suggests a crucial role in the cp1 swing and domain-domain interaction. here, the ... | 2014 | 25051973 |
| evidence for a hexaheteromeric methylenetetrahydrofolate reductase in moorella thermoacetica. | moorella thermoacetica can grow with h₂ and co₂, forming acetic acid from 2 co₂ via the wood-ljungdahl pathway. all enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (metf). we report here that the m. thermoacetica gene that putatively encodes this enzyme, metf, is part of a transcription unit also containing the genes hdrcba, mvhd, and metv. metf copurified with the other five proteins encoded in the unit in a hexaheteromeric comple ... | 2014 | 25002540 |
| acetyl coenzyme a synthetase is acetylated on multiple lysine residues by a protein acetyltransferase with a single gcn5-type n-acetyltransferase (gnat) domain in saccharopolyspora erythraea. | reversible lysine acetylation (rla) is used by cells of all domains of life to modulate protein function. to date, bacterial acetylation/deacetylation systems have been studied in a few bacteria (e.g., salmonella enterica, bacillus subtilis, escherichia coli, erwinia amylovora, mycobacterium tuberculosis, and geobacillus kaustophilus), but little is known about rla in antibiotic-producing actinomycetes. here, we identify the gcn5-like protein acetyltransferase acua of saccharopolyspora erythraea ... | 2014 | 24957627 |
| reco protein initiates dna recombination and strand annealing through two alternative dna binding mechanisms. | recombination mediator proteins (rmps) are important for genome stability in all organisms. several rmps support two alternative reactions: initiation of homologous recombination and dna annealing. we examined mechanisms of rmps in both reactions with mycobacterium smegmatis reco (msreco) and demonstrated that msreco interacts with ssdna by two distinct mechanisms. zinc stimulates msreco binding to ssdna during annealing, whereas the recombination function is zinc-independent and is regulated by ... | 2014 | 25170075 |
| crystal structure of a conserved hypothetical protein mj0927 from methanocaldococcus jannaschii reveals a novel quaternary assembly in the nif3 family. | a nif3 family protein of methanocaldococcus jannaschii, mj0927, is highly conserved from bacteria to humans. although several structures of bacterial nif3 proteins are known, no structure representing archaeal nif3 has yet been reported. the crystal structure of methanocaldococcus jannaschii mj0927 was determined at 2.47 å resolution to understand the structural differences between the bacterial and archaeal nif3 proteins. intriguingly, mj0927 is found to adopt an unusual assembly comprising a t ... | 2014 | 25243119 |
| evolution of transfer rna and the origin of the translation system. | | 2014 | 25221573 |
| escherichia coli d-malate dehydrogenase, a generalist enzyme active in the leucine biosynthesis pathway. | the enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of d-malate-based substrates with various specificities. here, we show that, in addition to its natural function affording bacterial growth on d-malate as a carbon source, the d-malate dehydrogenase of escherichia coli (ecdmla) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leub(-) strain lacking the paralogous isopropylmalate dehydrogenase enzy ... | 2014 | 25160617 |
| interplay between oxygen and fe-s cluster biogenesis: insights from the suf pathway. | iron-sulfur (fe-s) cluster metalloproteins conduct essential functions in nearly all contemporary forms of life. the nearly ubiquitous presence of fe-s clusters and the fundamental requirement for fe-s clusters in both aerobic and anaerobic archaea, bacteria, and eukarya suggest that these clusters were likely integrated into central metabolic pathways early in the evolution of life prior to the widespread oxidation of earth's atmosphere. intriguingly, fe-s cluster-dependent metabolism is sensit ... | 2014 | 25153801 |
| how lamina-associated polypeptide 1 (lap1) activates torsin. | lamina-associated polypeptide 1 (lap1) resides at the nuclear envelope and interacts with torsins, poorly understood endoplasmic reticulum (er)-localized aaa+ atpases, through a conserved, perinuclear domain. we determined the crystal structure of the perinuclear domain of human lap1. lap1 possesses an atypical aaa+ fold. while lap1 lacks canonical nucleotide binding motifs, its strictly conserved arginine 563 is positioned exactly where the arginine finger of canonical aaa+ atpases is found. ba ... | 2014 | 25149450 |
| crisprstrand: predicting repeat orientations to determine the crrna-encoding strand at crispr loci. | the discovery of crispr-cas systems almost 20 years ago rapidly changed our perception of the bacterial and archaeal immune systems. crispr loci consist of several repetitive dna sequences called repeats, inter-spaced by stretches of variable length sequences called spacers. this crispr array is transcribed and processed into multiple mature rna species (crrnas). a single crrna is integrated into an interference complex, together with crispr-associated (cas) proteins, to bind and degrade invadin ... | 2014 | 25161238 |
| dna binding properties of the small cascade subunit csa5. | crispr-cas systems provide immunity against viral attacks in archaeal and bacterial cells. type i systems employ a cas protein complex termed cascade, which utilizes small crispr rnas to detect and degrade the exogenic dna. a small sequence motif, the pam, marks the foreign substrates. previously, a recombinant type i-a cascade complex from the archaeon thermoproteus tenax was shown to target and degrade dna in vitro, dependent on a native pam sequence. here, we present the biochemical analysis ... | 2014 | 25148031 |
| comparison of histidine recognition in human and trypanosomatid histidyl-trna synthetases. | as part of a project aimed at obtaining selective inhibitors and drug-like compounds targeting trna synthetases from trypanosomatids, we have elucidated the crystal structure of human cytosolic histidyl-trna synthetase (hs-chisrs) in complex with histidine in order to be able to compare human and parasite enzymes. the resultant structure of hs-chisrs•his represents the substrate-bound state (h-state) of the enzyme. it provides an interesting opportunity to compare with ligand-free and imidazole- ... | 2014 | 25151410 |
| dissection of structural and functional requirements that underlie the interaction of erdj3 protein with substrates in the endoplasmic reticulum. | erdj3, a mammalian endoplasmic reticulum (er) hsp40/dnaj family member, binds unfolded proteins, transfers them to bip, and concomitantly stimulates bip atpase activity. however, the requirements for erdj3 binding to and release from substrates in cells are not well understood. we found that erdj3 homodimers that cannot stimulate the atpase activity of bip (qpd mutants) bound to unfolded er proteins under steady state conditions in much greater amounts than wild-type erdj3. this was due to reduc ... | 2014 | 25143379 |
| investigating the thermostability of succinate: quinone oxidoreductase enzymes by direct electrochemistry at swnts-modified electrodes and ftir spectroscopy. | succinate: quinone reductases (sqrs) are the enzymes that couple the oxidation of succinate and the reduction of quinones in the respiratory chain of prokaryotes and eukaryotes. herein, we compare the temperature-dependent activity and structural stability of two sqrs, the first from the mesophilic bacterium escherichia coli and the second from the thermophilic bacterium thermus thermophilus, using a combined electrochemical and infrared spectroscopy approach. direct electron transfer was achiev ... | 2014 | 25139263 |
| structure analysis of free and bound states of an rna aptamer against ribosomal protein s8 from bacillus anthracis. | several protein-targeted rna aptamers have been identified for a variety of applications and although the affinities of numerous protein-aptamer complexes have been determined, the structural details of these complexes have not been widely explored. we examined the structural accommodation of an rna aptamer that binds bacterial r-protein s8. the core of the primary binding site for s8 on helix 21 of 16s rrna contains a pair of conserved base triples that mold the sugar-phosphate backbone to s8. ... | 2014 | 25140011 |
| formation and structures of groel:groes2 chaperonin footballs, the protein-folding functional form. | the groe chaperonins assist substrate protein (sp) folding by cycling through several conformational states. with each cycle the sp is, in turn, captured, unfolded, briefly encapsulated (t1/2 ∼ 1 s), and released by the chaperonin complex. the protein-folding functional form is the us-football-shaped groel:groes2 complex. we report structures of two such "football" complexes to ∼ 3.7-å resolution; one is empty whereas the other contains encapsulated sp in both chambers. although encapsulated sp ... | 2014 | 25136110 |
| a proton wire to couple aminoacyl-trna accommodation and peptide-bond formation on the ribosome. | during peptide-bond formation on the ribosome, the α-amine of an aminoacyl-trna attacks the ester carbonyl carbon of a peptidyl-trna to yield a peptide lengthened by one amino acid. although the ribosome's contribution to catalysis is predominantly entropic, the lack of high-resolution structural data for the complete active site in complex with full-length ligands has made it difficult to assess how the ribosome might influence the pathway of the reaction. here, we present crystal structures of ... | 2014 | 25132179 |
| total synthesis of viridicatumtoxin b and analogues thereof: strategy evolution, structural revision, and biological evaluation. | the details of the total synthesis of viridicatumtoxin b (1) are described. initial synthetic strategies toward this intriguing tetracycline antibiotic resulted in the development of key alkylation and lewis acid-mediated spirocyclization reactions to form the hindered ef spirojunction, as well as michael-dieckmann reactions to set the a and c rings. the use of an aromatic a-ring substrate, however, was found to be unsuitable for the introduction of the requisite hydroxyl groups at carbons 4a an ... | 2014 | 25317739 |
| structural insights into +1 frameshifting promoted by expanded or modification-deficient anticodon stem loops. | maintenance of the correct reading frame on the ribosome is essential for accurate protein synthesis. here, we report structures of the 70s ribosome bound to frameshift suppressor trna(sufa6) and n1-methylguanosine at position 37 (m(1)g37) modification-deficient anticodon stem loop(pro), both of which cause the ribosome to decode 4 rather than 3 nucleotides, resulting in a +1 reading frame. our results reveal that decoding at +1 suppressible codons causes suppressor trna(sufa6) to undergo a rear ... | 2014 | 25128388 |
| structural biology. crystal structure of a crispr rna-guided surveillance complex bound to a ssdna target. | in prokaryotes, rna derived from type i and type iii crispr loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. in escherichia coli, this 405-kilodalton complex is called cascade. we report the crystal structure of cascade bound to a single-stranded dna (ssdna) target at a resolution of 3.03 angstroms. the structure reveals that the crispr rna and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. this noncan ... | 2014 | 25123481 |
| dead-box rna helicase domains exhibit a continuum between complete functional independence and high thermodynamic coupling in nucleotide and rna duplex recognition. | dead-box helicases catalyze the non-processive unwinding of double-stranded rna (dsrna) at the expense of adenosine triphosphate (atp) hydrolysis. nucleotide and rna binding and unwinding are mediated by the reca domains of the helicase core, but their cooperation in these processes remains poorly understood. we therefore investigated dsrna and nucleotide binding by the helicase cores and the isolated n- and c-terminal reca domains (reca_n, reca_c) of the dead-box proteins hera and yxin by stead ... | 2014 | 25123660 |
| allosteric regulation of a protein acetyltransferase in micromonospora aurantiaca by the amino acids cysteine and arginine. | act domains (amino acid-binding domains) are linked to a wide range of metabolic enzymes that are regulated by amino acid concentration. seventy proteins with act-gcn5-related n-acetyltransferase (gnat) domain organization were found in actinomycetales. in this study, we investigate the act-containing gnat acetyltransferase, micau_1670 (makat), from micromonospora aurantiaca atcc 27029. arginine and cysteine were identified as ligands by monitoring the conformational changes that occur upon amin ... | 2014 | 25124041 |
| discovery of a eukaryotic pyrroloquinoline quinone-dependent oxidoreductase belonging to a new auxiliary activity family in the database of carbohydrate-active enzymes. | pyrroloquinoline quinone (pqq) is a redox cofactor utilized by a number of prokaryotic dehydrogenases. not all prokaryotic organisms are capable of synthesizing pqq, even though it plays important roles in the growth and development of many organisms, including humans. the existence of pqq-dependent enzymes in eukaryotes has been suggested based on homology studies or the presence of pqq-binding motifs, but there has been no evidence that such enzymes utilize pqq as a redox cofactor. however, du ... | 2014 | 25121592 |
| dna binding polarity, dimerization, and atpase ring remodeling in the cmg helicase of the eukaryotic replisome. | the cdc45/mcm2-7/gins (cmg) helicase separates dna strands during replication in eukaryotes. how the cmg is assembled and engages dna substrates remains unclear. using electron microscopy, we have determined the structure of the cmg in the presence of atpγs and a dna duplex bearing a 3' single-stranded tail. the structure shows that the mcm subunits of the cmg bind preferentially to single-stranded dna, establishes the polarity by which dna enters into the mcm2-7 pore, and explains how cdc45 hel ... | 2014 | 25117490 |