| x-ray structures of two proteins belonging to pfam duf178 revealed unexpected structural similarity to the duf191 pfam family. | pfam is a comprehensive collection of protein domains and families, with a range of well-established information including genome annotation. pfam has two large series of functionally uncharacterized families, known as domains of unknown function (dufs) and uncharacterized protein families (upfs). | 2007 | 17908300 |
| cocrystallizing natural rna with its unnatural mirror image: biochemical and preliminary x-ray diffraction analysis of a 5s rrna a-helix racemate. | chemically synthesized rnas with the unnatural l-configuration possess enhanced in vivo stability and nuclease resistance, which is a highly desirable property for pharmacological applications. for a structural comparison, both l- and d-rna oligonucleotides of a shortened thermus flavus 5s rrna a-helix were chemically synthesized. the enantiomeric rna duplexes were stochiometrically cocrystallized as a racemate, which enabled analysis of the d- and l-rna enantiomers in the same crystals. in addi ... | 2007 | 17909284 |
| human trna(gly) acceptor-stem microhelix: crystallization and preliminary x-ray diffraction analysis at 1.2 a resolution. | the major dissimilarities between the eukaryotic/archaebacterial-type and eubacterial-type glycyl-trna synthetase systems (glyrs; class ii aminoacyl-trna synthetases) represent an intriguing example of evolutionarily divergent solutions to similar biological functions. the differences in the identity elements of the respective trna(gly) systems are located within the acceptor stem and include the discriminator base u73. in the present work, the human trna(gly) acceptor-stem microhelix was crysta ... | 2007 | 17909289 |
| functional smpb-ribosome interactions require tmrna. | small protein b (smpb) is a requisite component of the transfer messenger rna (tmrna)-mediated bacterial translational quality control system known as trans-translation. the initial binding of tmrna and its subsequent accommodation into the ribosomal a-site are activities intimately linked to smpb protein function. from a mechanistic perspective, two key unanswered questions that require further investigation are: 1) what constitutes a stalled ribosome recognition complex and 2) does smpb pre-bi ... | 2007 | 17911096 |
| analysis of gene order data supports vertical inheritance of the leukotoxin operon and genome rearrangements in the 5' flanking region in genus mannheimia. | the mannheimia subclades belong to the same bacterial genus, but have taken divergent paths toward their distinct lifestyles. for example, m. haemolytica + m. glucosida are potential pathogens of the respiratory tract in the mammalian suborder ruminantia, whereas m. ruminalis, the supposed sister group, lives as a commensal in the ovine rumen. we have tested the hypothesis that vertical inheritance of the leukotoxin (lktcabd) operon has occurred from the last common ancestor of genus mannheimia ... | 2007 | 17915007 |
| the carboxy-terminal coiled-coil of the rna polymerase beta'-subunit is the main binding site for gre factors. | bacterial gre transcript cleavage factors stimulate the intrinsic endonucleolytic activity of rna polymerase (rnap) to rescue stalled transcription complexes. they bind to rnap and extend their coiled-coil (cc) domains to the catalytic centre through the secondary channel. three existing models for the gre-rnap complex postulate congruent mechanisms of gre-assisted catalysis, while offering conflicting views of the gre-rnap interactions. here, we report the greb structure of escherichia coli. th ... | 2007 | 17917675 |
| evolution and functional characterization of the rh50 gene from the ammonia-oxidizing bacterium nitrosomonas europaea. | the family of ammonia and ammonium channel proteins comprises the amt proteins, which are present in all three domains of life with the notable exception of vertebrates, and the homologous rh proteins (rh50 and rh30) that have been described thus far only in eukaryotes. the existence of an rh50 gene in bacteria was first revealed by the genome sequencing of the ammonia-oxidizing bacterium nitrosomonas europaea. here we have used a phylogenetic approach to study the evolution of the n. europaea r ... | 2007 | 17921289 |
| orotate phosphoribosyltransferase from corynebacterium ammoniagenes lacking a conserved lysine. | the pyre gene, encoding orotate phosphoribosyltransferase (oprtase), was cloned by nested pcr and colony blotting from corynebacterium ammoniagenes atcc 6872, which is widely used in nucleotide production. sequence analysis shows that there is a lack of an important conserved lysine (lys 73 in salmonella enterica serovar typhimurium oprtase) in the c. ammoniagenes oprtase. this lysine has been considered to contribute to the initiation of catalysis. the enzyme was overexpressed and purified from ... | 2007 | 17921291 |
| insights into hsp70 chaperone activity from a crystal structure of the yeast hsp110 sse1. | classic hsp70 chaperones assist in diverse processes of protein folding and translocation, and hsp110s had seemed by sequence to be distant relatives within an hsp70 superfamily. the 2.4 a resolution structure of sse1 with atp shows that hsp110s are indeed hsp70 relatives, and it provides insight into allosteric coupling between sites for atp and polypeptide-substrate binding in hsp70s. subdomain structures are similar in intact sse1(atp) and in the separate hsp70 domains, but conformational dis ... | 2007 | 17923091 |
| genetic and phenotypic identification of fusidic acid-resistant mutants with the small-colony-variant phenotype in staphylococcus aureus. | small-colony variants (scvs) of staphylococcus aureus are a slow-growing subpopulation whose phenotypes can include resistance to aminoglycosides, defects in electron transport, and enhanced persistence in mammalian cells. here we show that a subset of mutants selected as scvs by reduced susceptibility to aminoglycosides are resistant to the antibiotic fusidic acid (fa) and conversely that a subset of mutants selected for resistance to fa are scvs. mutation analysis reveals different genetic cla ... | 2007 | 17923494 |
| a cysteine-rich ccg domain contains a novel [4fe-4s] cluster binding motif as deduced from studies with subunit b of heterodisulfide reductase from methanothermobacter marburgensis. | heterodisulfide reductase (hdr) of methanogenic archaea with its active-site [4fe-4s] cluster catalyzes the reversible reduction of the heterodisulfide (com-s-s-cob) of the methanogenic coenzyme m (com-sh) and coenzyme b (cob-sh). com-hdr, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with com-sh, is a novel type of [4fe-4s]3+ cluster with com-sh as a ligand. subunit hdrb of the methanothermobacter marburgensis hdrabc holoenzyme contains two cy ... | 2007 | 17929940 |
| interactions and dynamics of the shine dalgarno helix in the 70s ribosome. | the crystal structure of an initiation-like 70s ribosome complex containing an 8-bp shine-dalgarno (sd) helix was determined at 3.8-a resolution. translation-libration-screw analysis showed that the inherent anisotropic motions of the sd helix were biased along its helical axis, suggesting that during the first step of translocation, the sd helix moves along its helical screw axis. contacts between the sd helix and the ribosome were primarily through interactions with helices 23a, 26, and 28 of ... | 2007 | 17940016 |
| the wobble hypothesis revisited: uridine-5-oxyacetic acid is critical for reading of g-ending codons. | according to crick's wobble hypothesis, trnas with uridine at the wobble position (position 34) recognize a- and g-, but not u- or c-ending codons. however, u in the wobble position is almost always modified, and salmonella enterica trnas containing the modified nucleoside uridine-5-oxyacetic acid (cmo(5)u34) at this position are predicted to recognize u- (but not c-) ending codons, in addition to a- and g-ending codons. we have constructed a set of s. enterica mutants with only the cmo(5)u-cont ... | 2007 | 17942742 |
| the 3d rrna modification maps database: with interactive tools for ribosome analysis. | the 3d rrna modification maps database is the first general resource of information about the locations of modified nucleotides within the 3d structure of the full ribosome, with mrna and trnas in the a-, p- and e-sites. the database supports analyses for several model organisms, including higher eukaryotes, and enables users to construct 3d maps for other organisms. data are provided for human and plant (arabidopsis) ribosomes, and for other representative organisms from eubacteria, archaea and ... | 2008 | 17947322 |
| the 3d rrna modification maps database: with interactive tools for ribosome analysis. | the 3d rrna modification maps database is the first general resource of information about the locations of modified nucleotides within the 3d structure of the full ribosome, with mrna and trnas in the a-, p- and e-sites. the database supports analyses for several model organisms, including higher eukaryotes, and enables users to construct 3d maps for other organisms. data are provided for human and plant (arabidopsis) ribosomes, and for other representative organisms from eubacteria, archaea and ... | 2008 | 17947322 |
| the structure of mycobacteria 2c-methyl-d-erythritol-2,4-cyclodiphosphate synthase, an essential enzyme, provides a platform for drug discovery. | the prevalence of tuberculosis, the prolonged and expensive treatment that this disease requires and an increase in drug resistance indicate an urgent need for new treatments. the 1-deoxy-d-xylulose 5-phosphate pathway of isoprenoid precursor biosynthesis is an attractive chemotherapeutic target because it occurs in many pathogens, including mycobacterium tuberculosis, and is absent from humans. to underpin future drug development it is important to assess which enzymes in this biosynthetic path ... | 2007 | 17956607 |
| interaction of smpb with ribosome from directed hydroxyl radical probing. | to add a tag-peptide for degradation to the nascent polypeptide in a stalled ribosome, an unusual translation called trans-translation is facilitated by transfer-messenger rna (tmrna) having an upper half of the trna structure and the sequence encoding the tag-peptide except the first alanine. during this event, tmrna enters the vacant a-site of the stalled ribosome without a codon-anticodon interaction, but with a protein factor smpb. here, we studied the sites and modes of binding of smpb to t ... | 2007 | 17959652 |
| domain i of ribosomal protein l1 is sufficient for specific rna binding. | ribosomal protein l1 has a dual function as a ribosomal protein binding 23s rrna and as a translational repressor binding its mrna. l1 is a two-domain protein with n- and c-termini located in domain i. earlier it was shown that l1 interacts with the same targets on both rrna and mrna mainly through domain i. we have suggested that domain i is necessary and sufficient for specific rna-binding by l1. to test this hypothesis, a truncation mutant of l1 from thermus thermophilus, representing domain ... | 2007 | 17962298 |
| mutational analysis of s12 protein and implications for the accuracy of decoding by the ribosome. | the fidelity of aminoacyl-trna selection by the ribosome depends on a conformational switch in the decoding center of the small ribosomal subunit induced by cognate but not by near-cognate aminoacyl-trna. the aminoglycosides paromomycin and streptomycin bind to the decoding center and induce related structural rearrangements that explain their observed effects on miscoding. structural and biochemical studies have identified ribosomal protein s12 (as well as specific nucleotides in 16s ribosomal ... | 2007 | 17967466 |
| novel hexamerization motif is discovered in a conserved cytoplasmic protein from salmonella typhimurium. | the cytoplasmic protein stm3548 of unknown function obtained from a strain of salmonella typhimurium was determined by x-ray crystallography at a resolution of 2.25 a. the asymmetric unit contains a hexamer of structurally identical monomers. the monomer is a globular domain with a long beta-hairpin protrusion that distinguishes this structure. this beta-hairpin occupies a central position in the hexamer, and its residues participate in the majority of interactions between subunits of the hexame ... | 2007 | 17968677 |
| translation initiation region sequence preferences in escherichia coli. | the mrna translation initiation region (tir) comprises the initiator codon, shine-dalgarno (sd) sequence and translational enhancers. probably the most abundant class of enhancers contains a/u-rich sequences. we have tested the influence of sd sequence length and the presence of enhancers on the efficiency of translation initiation. | 2007 | 17973990 |
| zinc is the metal cofactor of borrelia burgdorferi peptide deformylase. | peptide deformylase (pdf, e.c. 3.5.1.88) catalyzes the removal of n-terminal formyl groups from nascent ribosome-synthesized polypeptides. pdf contains a catalytically essential divalent metal ion, which is tetrahedrally coordinated by three protein ligands (his, his, and cys) and a water molecule. previous studies revealed that the metal cofactor is a fe2+ ion in escherichia coli and many other bacterial pdfs. in this work, we found that pdfs from two iron-deficient bacteria, borrelia burgdorfe ... | 2007 | 17977509 |
| the role of the mitochondrial glycine cleavage complex in the metabolism and virulence of the protozoan parasite leishmania major. | for the human pathogen leishmania major, a key metabolic function is the synthesis of thymidylate, which requires 5,10-methylenetetrahydrofolate (5,10-ch(2)-thf). 5,10-ch(2)-thf can be synthesized from glycine by the mitochondrial glycine cleavage complex (gcc). bioinformatic analysis revealed the four subunits of the gcc in the l. major genome, and the role of the gcc in parasite metabolism and virulence was assessed through studies of the p subunit (glycine decarboxylase (gcvp)). first, a tagg ... | 2008 | 17981801 |
| the role of the mitochondrial glycine cleavage complex in the metabolism and virulence of the protozoan parasite leishmania major. | for the human pathogen leishmania major, a key metabolic function is the synthesis of thymidylate, which requires 5,10-methylenetetrahydrofolate (5,10-ch(2)-thf). 5,10-ch(2)-thf can be synthesized from glycine by the mitochondrial glycine cleavage complex (gcc). bioinformatic analysis revealed the four subunits of the gcc in the l. major genome, and the role of the gcc in parasite metabolism and virulence was assessed through studies of the p subunit (glycine decarboxylase (gcvp)). first, a tagg ... | 2008 | 17981801 |
| structural basis of j cochaperone binding and regulation of hsp70. | the many protein processing reactions of the atp-hydrolyzing hsp70s are regulated by j cochaperones, which contain j domains that stimulate hsp70 atpase activity and accessory domains that present protein substrates to hsp70s. we report the structure of a j domain complexed with a j responsive portion of a mammalian hsp70. the j domain activates atpase activity by directing the linker that connects the hsp70 nucleotide binding domain (nbd) and substrate binding domain (sbd) toward a hydrophobic ... | 2007 | 17996706 |
| structural aspects of rbfa action during small ribosomal subunit assembly. | ribosome binding factor a (rbfa) is a bacterial cold shock response protein, required for an efficient processing of the 5' end of the 16s ribosomal rna (rrna) during assembly of the small (30s) ribosomal subunit. here we present a crystal structure of thermus thermophilus (tth) rbfa and a three-dimensional cryo-electron microscopic (em) map of the tth 30s*rbfa complex. rbfa binds to the 30s subunit in a position overlapping the binding sites of the a and p site trnas, and rbfa's functionally im ... | 2007 | 17996707 |
| two distinct components of release factor function uncovered by nucleophile partitioning analysis. | during translation termination, release factor (rf) protein catalyzes a hydrolytic reaction in the large subunit peptidyl transferase center to release the finished polypeptide chain. while the mechanism of catalysis of peptide release remains obscure, important contributing factors have been identified, including conserved active-site nucleotides and a ggq tripeptide motif in the rf. here we describe pre-steady-state kinetic and nucleophile competition experiments to examine rf contributions to ... | 2007 | 17996709 |
| fourier transform infrared characterization of a cub-nitrosyl complex in cytochrome ba3 from thermus thermophilus: relevance to no reductase activity in heme-copper terminal oxidases. | the two heme-copper terminal oxidases of thermus thermophilus have been shown to catalyze the two-electron reduction of nitric oxide (no) to nitrous oxide (n2o) [giuffre, a.; stubauer, g.; sarti, p.; brunori, m.; zumft, w. g.; buse, g.; soulimane, t. proc. natl. acad. sci. u.s.a. 1999, 96, 14718-14723]. while it is well-established that no binds to the reduced heme a3 to form a low-spin heme {feno}7 species, the role cub plays in the binding of the second no remains unclear. here we present low- ... | 2007 | 17997553 |
| structural basis of a ribozyme's thermostability: p1-l9 interdomain interaction in rnase p rna. | for stability, many catalytic rnas rely on long-range tertiary interactions, the precise role of each often being unclear. here we demonstrate that one of the three interdomain architectural struts of rnase p rna (p rna) is the key to activity at higher temperatures: disrupting the p1-l9 helix-tetraloop interaction in p rna of the thermophile thermus thermophilus decreased activity at high temperatures in the rna-alone reaction and at low mg2+ concentrations in the holoenzyme reaction. conversel ... | 2008 | 17998289 |
| a novel chromatography system to isolate active ribosomes from pathogenic bacteria. | we have developed a novel chromatography for the rapid isolation of active ribosomes from bacteria without the use of harsh conditions or lengthy procedures that damage ribosomes. ribosomes interact with an alkyl linker attached to the resin, apparently through their rna component. examples are given with ribosomes from escherichia coli, deinococcus radiodurans, and with clinical isolates of streptococcus pneumoniae and methicillin-resistant staphylococcus aureus (mrsa). the ribosomes obtained b ... | 2008 | 17998293 |
| the process of displacing the single-stranded dna-binding protein from single-stranded dna by reco and recr proteins. | the regions of single-stranded (ss) dna that result from dna damage are immediately coated by the ssdna-binding protein (ssb). recf pathway proteins facilitate the displacement of ssb from ssdna, allowing the reca protein to form protein filaments on the ssdna region, which facilitates the process of recombinational dna repair. in this study, we examined the mechanism of ssb displacement from ssdna using purified thermus thermophilus recf pathway proteins. to date, reco and recr are thought to a ... | 2008 | 18000001 |
| eukaryotic ribosomal rna determinants of aminoglycoside resistance and their role in translational fidelity. | recent studies of prokaryotic ribosomes have dramatically increased our knowledge of ribosomal rna (rrna) structure, functional centers, and their interactions with antibiotics. however, much less is known about how rrna function differs between prokaryotic and eukaryotic ribosomes. the core decoding sites are identical in yeast and human 18s rrnas, suggesting that insights obtained in studies with yeast rrna mutants can provide information about ribosome function in both species. in this study, ... | 2008 | 18003936 |
| cloning, expression, purification, crystallization and preliminary x-ray diffraction analysis of universal stress protein f (ynaf) from salmonella typhimurium. | the universal stress protein uspf (ynaf) is a small cytoplasmic bacterial protein. the expression of stress proteins is enhanced when cells are exposed to heat shock, nutrition starvation and certain other stress-inducing agents. ynaf promotes cell survival during prolonged exposure to stress and may activate a general mechanism for stress endurance. this manuscript reports preliminary crystallographic studies on ynaf from salmonella typhimurium. the gene coding for ynaf was cloned and overexpre ... | 2007 | 18007050 |
| crystallization and preliminary x-ray diffraction studies of tetrameric malate dehydrogenase from the novel antarctic psychrophile flavobacterium frigidimaris kuc-1. | flavobacterium frigidimaris kuc-1 is a novel psychrotolerant bacterium isolated from antarctic seawater. malate dehydrogenase (mdh) is an essential metabolic enzyme in the citric acid cycle and has been cloned, overexpressed and purified from f. frigidimaris kuc-1. in contrast to the already known dimeric form of mdh from the psychrophile aquaspirillium arcticum, f. frigidimaris mdh exists as a tetramer. it was crystallized at 288 k by the hanging-drop vapour-diffusion method using ammonium sulf ... | 2007 | 18007057 |
| the methyltransferase yfgb/rlmn is responsible for modification of adenosine 2503 in 23s rrna. | a2503 in 23s rrna of the gram-negative bacterium escherichia coli is located in a functionally important region of the ribosome, at the entrance to the nascent peptide exit tunnel. in e. coli, and likely in other species, this adenosine residue is post-transcriptionally modified to m2a. the enzyme responsible for this modification was previously unknown. we identified e. coli protein yfgb, which belongs to the radical sam enzyme superfamily, as the methyltransferase that modifies a2503 of 23s rr ... | 2008 | 18025251 |
| pseudouridylation of helix 69 of 23s rrna is necessary for an effective translation termination. | escherichia coli strains with inactivated rlud genes were previously found to lack the conserved pseudouridines in helix 69 of 23s ribosomal rna and to grow slowly. a suppressor mutant was isolated with a near normal growth rate that had changed the conserved glu-172 codon to a lys codon in prfb, encoding translation termination factor rf2. when nonsense suppression in strains with all combinations of prfb(+)/prfb(e172k) and rlud(+)/rlud::cat was analyzed, misreading of all three stop codons as ... | 2007 | 18032607 |
| the stator complex of the a1a0-atp synthase--structural characterization of the e and h subunits. | archaeal atp synthase (a-atpase) is the functional homolog to the atp synthase found in bacteria, mitochondria and chloroplasts, but the enzyme is structurally more related to the proton-pumping vacuolar atpase found in the endomembrane system of eukaryotes. we have cloned, overexpressed and characterized the stator-forming subunits e and h of the a-atpase from the thermoacidophilic archaeon, thermoplasma acidophilum. size exclusion chromatography, cd, matrix-assisted laser desorption ionization ... | 2008 | 18036615 |
| the stator complex of the a1a0-atp synthase--structural characterization of the e and h subunits. | archaeal atp synthase (a-atpase) is the functional homolog to the atp synthase found in bacteria, mitochondria and chloroplasts, but the enzyme is structurally more related to the proton-pumping vacuolar atpase found in the endomembrane system of eukaryotes. we have cloned, overexpressed and characterized the stator-forming subunits e and h of the a-atpase from the thermoacidophilic archaeon, thermoplasma acidophilum. size exclusion chromatography, cd, matrix-assisted laser desorption ionization ... | 2008 | 18036615 |
| reversible dissociation of flavin mononucleotide from the mammalian membrane-bound nadh: ubiquinone oxidoreductase (complex i). | conditions for the reversible dissociation of flavin mononucleotide (fmn) from the membrane-bound mitochondrial nadh:ubiquinone oxidoreductase (complex i) are described. the catalytic activities of the enzyme, i.e. rotenone-insensitive nadh:hexaammineruthenium iii reductase and rotenone-sensitive nadh:quinone reductase decline when bovine heart submitochondrial particles are incubated with nadh in the presence of rotenone or cyanide at alkaline ph. fmn protects and fully restores the nadh-induce ... | 2007 | 18037377 |
| structural basis for different substrate specificities of two adp-ribose pyrophosphatases from thermus thermophilus hb8. | adp-ribose (adpr) is one of the main substrates of nudix proteins. among the eight nudix proteins of thermus thermophilus hb8, we previously determined the crystal structure of ndx4, an adpr pyrophosphatase (adprase). in this study we show that ndx2 of t. thermophilus also preferentially hydrolyzes adpr and flavin adenine dinucleotide and have determined its crystal structure. we have determined the structures of ndx2 alone and in complex with mg2+, with mg2+ and amp, and with mg2+ and a nonhydr ... | 2008 | 18039767 |
| discrimination of class i cyclobutane pyrimidine dimer photolyase from blue light photoreceptors by single methionine residue. | dna photolyase recognizes ultraviolet-damaged dna and breaks improperly formed covalent bonds within the cyclobutane pyrimidine dimer by a light-activated electron transfer reaction between the flavin adenine dinucleotide, the electron donor, and cyclobutane pyrimidine dimer, the electron acceptor. theoretical analysis of the electron-tunneling pathways of the dna photolyase derived from anacystis nidulans can reveal the active role of the protein environment in the electron transfer reaction. h ... | 2008 | 18055535 |
| discrimination of class i cyclobutane pyrimidine dimer photolyase from blue light photoreceptors by single methionine residue. | dna photolyase recognizes ultraviolet-damaged dna and breaks improperly formed covalent bonds within the cyclobutane pyrimidine dimer by a light-activated electron transfer reaction between the flavin adenine dinucleotide, the electron donor, and cyclobutane pyrimidine dimer, the electron acceptor. theoretical analysis of the electron-tunneling pathways of the dna photolyase derived from anacystis nidulans can reveal the active role of the protein environment in the electron transfer reaction. h ... | 2008 | 18055535 |
| redox properties of thermus thermophilus ba3: different electron-proton coupling in oxygen reductases? | a comprehensive study of the thermodynamic redox behavior of the hemes of the ba3 enzyme from thermus thermophilus, a b-type heme-copper oxygen reductase, is presented. this enzyme, in contrast to those having a single type of heme, allows the b- and a-type hemes to be monitored separately by visible spectroscopy and the reduction potential of each heme to be determined unequivocally. the relative order of the midpoint reduction potentials of each center changed in the ph range from 6 to 8.4, an ... | 2008 | 18065462 |
| yeast as a model of human mitochondrial trna base substitutions: investigation of the molecular basis of respiratory defects. | we investigate the relationships between acylation defects and structure alterations due to base substitutions in yeast mitochondrial (mt) trna(uur)(leu). the studied substitutions are equivalent to the a3243g and t3250c human pathogenetic trna mutations. our data show that both mutations can produce trna(uur)(leu) acylation defects, although to a different extent. for mutant a14g (equivalent to melas a3243g base substitution), the presence of the trna and its defective aminoacylation could be o ... | 2008 | 18065717 |
| dodecamer rotor ring defines h+/atp ratio for atp synthesis of prokaryotic v-atpase from thermus thermophilus. | atp synthesis by v-atpase from the thermophilic bacterium thermus thermophilus driven by the acid-base transition was investigated. the rate of atp synthesis increased in parallel with the increase in proton motive force (pmf) >110 mv, which is composed of a difference in proton concentration (deltaph) and the electrical potential differences (deltapsi) across membranes. the optimum rate of synthesis reached 85 s(-1), and the h(+)/atp ratio of 4.0 +/- 0.1 was obtained. atp was synthesized at a c ... | 2007 | 18077374 |
| soj (para) dna binding is mediated by conserved arginines and is essential for plasmid segregation. | soj is a member of the para family of atpases involved in plasmid and chromosomal segregation. it binds nonspecifically and cooperatively to dna although the function of this binding is unknown. here, we show that mutation of conserved arginine residues that map to the surface of bacillus subtilis soj caused only minimal effects on nucleotide-dependent dimerization but had dramatic effects on dna binding. using a model plasmid partitioning system in escherichia coli, we find that soj dna-binding ... | 2007 | 18077387 |
| akap18 contains a phosphoesterase domain that binds amp. | protein kinase a anchoring proteins (akaps), defined by their capacity to target the camp-dependent protein kinase to distinct subcellular locations, function as molecular scaffolds mediating the assembly of multicomponent complexes to integrate and organise multiple signalling events. despite their central importance in regulating cellular processes, little is known regarding their diverse structures and molecular mechanisms. here, using bioinformatics and x-ray crystallography, we define a cen ... | 2008 | 18082768 |
| akap18 contains a phosphoesterase domain that binds amp. | protein kinase a anchoring proteins (akaps), defined by their capacity to target the camp-dependent protein kinase to distinct subcellular locations, function as molecular scaffolds mediating the assembly of multicomponent complexes to integrate and organise multiple signalling events. despite their central importance in regulating cellular processes, little is known regarding their diverse structures and molecular mechanisms. here, using bioinformatics and x-ray crystallography, we define a cen ... | 2008 | 18082768 |
| escherichia coli cytosolic glycerophosphodiester phosphodiesterase (ugpq) requires mg2+, co2+, or mn2+ for its enzyme activity. | escherichia coli cytosolic glycerophosphodiester phosphodiesterase, ugpq, functions in the absence of other proteins encoded by the ugp operon and requires mg2+, mn2+, or co2+, in contrast to ca2+-dependent periplasmic glycerophosphodiester phosphodiesterase, glpq. ugpq has broad substrate specificity toward various glycerophosphodiesters, producing sn-glycerol-3-phosphate and the corresponding alcohols. ugpq accumulates under conditions of phosphate starvation, suggesting that it allows the uti ... | 2008 | 18083802 |
| escherichia coli cytosolic glycerophosphodiester phosphodiesterase (ugpq) requires mg2+, co2+, or mn2+ for its enzyme activity. | escherichia coli cytosolic glycerophosphodiester phosphodiesterase, ugpq, functions in the absence of other proteins encoded by the ugp operon and requires mg2+, mn2+, or co2+, in contrast to ca2+-dependent periplasmic glycerophosphodiester phosphodiesterase, glpq. ugpq has broad substrate specificity toward various glycerophosphodiesters, producing sn-glycerol-3-phosphate and the corresponding alcohols. ugpq accumulates under conditions of phosphate starvation, suggesting that it allows the uti ... | 2008 | 18083802 |
| structure of the minimized alpha/beta-hydrolase fold protein from thermus thermophilus hb8. | the gene encoding ttha1544 is a singleton found in the thermus thermophilus hb8 genome and encodes a 131-amino-acid protein. the crystal structure of ttha1544 has been determined at 2.0 a resolution by the single-wavelength anomalous dispersion method in order to elucidate its function. there are two molecules in the asymmetric unit. each molecule consists of four alpha-helices and six beta-strands, with the beta-strands composing a central beta-sheet. a structural homology search revealed that ... | 2007 | 18084077 |
| structure of bacillus subtilis superoxide dismutase. | the soda gene of bacillus subtilis was expressed in escherichia coli, purified and crystallized. the crystal structure of mnsod was solved by molecular replacement with four dimers per asymmetric unit and refined to an r factor of 21.1% at 1.8 a resolution. the dimer structure is very similar to that of the related enzyme from b. anthracis. larger structural differences were observed with the human mnsod, which has one less helix in the helical domain and a longer loop between two beta-strands a ... | 2007 | 18084079 |
| an unexpected outcome of surface engineering an integral membrane protein: improved crystallization of cytochrome ba(3) from thermus thermophilus. | past work has shown that it is feasible to mutate surface residues of soluble proteins and to a lesser extent membrane proteins in order to improve their crystallization behavior. described here is a successful application of this approach to the integral membrane protein thermus thermophilus cytochrome ba(3) oxidase. two mutant forms of this enzyme (i-k258r and i-k258r/ii-e4q) were created in which symmetrical crystal contacts within crystals of wild-type enzyme were modified. these mutant prot ... | 2007 | 18084085 |
| mycobacterium tuberculosis cyp130: crystal structure, biophysical characterization, and interactions with antifungal azole drugs. | cyp130 is one of the 20 mycobacterium tuberculosis cytochrome p450 enzymes, only two of which, cyp51 and cyp121, have so far been studied as individually expressed proteins. here we characterize a third heterologously expressed m. tuberculosis cytochrome p450, cyp130, by uv-visible spectroscopy, isothermal titration calorimetry, and x-ray crystallography, including determination of the crystal structures of ligand-free and econazole-bound cyp130 at a resolution of 1.46 and 3.0a(,) respectively. ... | 2008 | 18089574 |
| mycobacterium tuberculosis cyp130: crystal structure, biophysical characterization, and interactions with antifungal azole drugs. | cyp130 is one of the 20 mycobacterium tuberculosis cytochrome p450 enzymes, only two of which, cyp51 and cyp121, have so far been studied as individually expressed proteins. here we characterize a third heterologously expressed m. tuberculosis cytochrome p450, cyp130, by uv-visible spectroscopy, isothermal titration calorimetry, and x-ray crystallography, including determination of the crystal structures of ligand-free and econazole-bound cyp130 at a resolution of 1.46 and 3.0a(,) respectively. ... | 2008 | 18089574 |
| mammalian 2',3' cyclic nucleotide phosphodiesterase (cnp) can function as a trna splicing enzyme in vivo. | yeast and plant trna splicing entails discrete healing and sealing steps catalyzed by a trna ligase that converts the 2',3' cyclic phosphate and 5'-oh termini of the broken trna exons to 3'-oh/2'-po4 and 5'-po4 ends, respectively, then joins the ends to yield a 2'-po4, 3'-5' phosphodiester splice junction. the junction 2'-po4 is removed by a trna phosphotransferase, tpt1. animal cells have two potential trna repair pathways: a yeast-like system plus a distinctive mechanism, also present in archa ... | 2008 | 18094118 |
| assembly of the 5' and 3' minor domains of 16s ribosomal rna as monitored by tethered probing from ribosomal protein s20. | the ribosomal protein (r-protein) s20 is a primary binding protein. as such, it interacts directly and independently with the 5' domain as well as the 3' minor domain of 16s ribosomal rna (rrna) in minimal particles and the fully assembled 30s subunit. the interactions observed between r-protein s20 and the 5' domain of 16s rrna are quite extensive, while those between r-protein s20 and the 3' minor domain are significantly more limited. in this study, directed hydroxyl radical probing mediated ... | 2008 | 18155048 |
| assembly of the 5' and 3' minor domains of 16s ribosomal rna as monitored by tethered probing from ribosomal protein s20. | the ribosomal protein (r-protein) s20 is a primary binding protein. as such, it interacts directly and independently with the 5' domain as well as the 3' minor domain of 16s ribosomal rna (rrna) in minimal particles and the fully assembled 30s subunit. the interactions observed between r-protein s20 and the 5' domain of 16s rrna are quite extensive, while those between r-protein s20 and the 3' minor domain are significantly more limited. in this study, directed hydroxyl radical probing mediated ... | 2008 | 18155048 |
| a flexible peptide tether controls accessibility of a unique c-terminal rna-binding domain in leucyl-trna synthetases. | a unique c-terminal domain extension is required by most leucyl-trna synthetases (leurs) for aminoacylation. in one exception, the enzymatic activity of yeast mitochondrial leurs is actually impeded by its own c-terminal domain. it was proposed that the yeast mitochondrial leurs has compromised its aminoacylation activity to some extent and adapted its c terminus for a second role in rna splicing, which is also essential. x-ray crystal structures of the leurs-trna complex show that the 60 residu ... | 2008 | 18155724 |
| a flexible peptide tether controls accessibility of a unique c-terminal rna-binding domain in leucyl-trna synthetases. | a unique c-terminal domain extension is required by most leucyl-trna synthetases (leurs) for aminoacylation. in one exception, the enzymatic activity of yeast mitochondrial leurs is actually impeded by its own c-terminal domain. it was proposed that the yeast mitochondrial leurs has compromised its aminoacylation activity to some extent and adapted its c terminus for a second role in rna splicing, which is also essential. x-ray crystal structures of the leurs-trna complex show that the 60 residu ... | 2008 | 18155724 |
| crystal structure of bacillus stearothermophilus uvra provides insight into atp-modulated dimerization, uvrb interaction, and dna binding. | the nucleotide excision repair pathway corrects many structurally unrelated dna lesions. damage recognition in bacteria is performed by uvra, a member of the abc atpase superfamily whose functional form is a dimer with four nucleotide-binding domains (nbds), two per protomer. in the 3.2 a structure of uvra from bacillus stearothermophilus, we observe that the nucleotide-binding sites are formed in an intramolecular fashion and are not at the dimer interface as is typically found in other abc atp ... | 2008 | 18158267 |
| crystal structure of bacillus stearothermophilus uvra provides insight into atp-modulated dimerization, uvrb interaction, and dna binding. | the nucleotide excision repair pathway corrects many structurally unrelated dna lesions. damage recognition in bacteria is performed by uvra, a member of the abc atpase superfamily whose functional form is a dimer with four nucleotide-binding domains (nbds), two per protomer. in the 3.2 a structure of uvra from bacillus stearothermophilus, we observe that the nucleotide-binding sites are formed in an intramolecular fashion and are not at the dimer interface as is typically found in other abc atp ... | 2008 | 18158267 |
| protein factors in pre-mrna 3'-end processing. | most eukaryotic mrna precursors (premrnas) must undergo extensive processing, including cleavage and polyadenylation at the 3'-end. processing at the 3'-end is controlled by sequence elements in the pre-mrna (cis elements) as well as protein factors. despite the seeming biochemical simplicity of the processing reactions, more than 14 proteins have been identified for the mammalian complex, and more than 20 proteins have been identified for the yeast complex. the 3'-end processing machinery also ... | 2008 | 18158581 |
| genome evolution and the emergence of fruiting body development in myxococcus xanthus. | lateral gene transfer (lgt) is thought to promote speciation in bacteria, though well-defined examples have not been put forward. | 2007 | 18159227 |
| fitness of streptococcus pneumoniae fluoroquinolone-resistant strains with topoisomerase iv recombinant genes. | the low prevalence of ciprofloxacin-resistant (cp r) streptococcus pneumoniae isolates carrying recombinant topoisomerase iv genes could be attributed to a fitness cost imposed by the horizontal transfer, which often implies the acquisition of larger-than-normal pare-parc intergenic regions. a study of the transcription of these genes and of the fitness cost for 24 isogenic cp r strains was performed. six first-level transformants were obtained either with pcr products containing the parc quinol ... | 2008 | 18160515 |
| fitness of streptococcus pneumoniae fluoroquinolone-resistant strains with topoisomerase iv recombinant genes. | the low prevalence of ciprofloxacin-resistant (cp r) streptococcus pneumoniae isolates carrying recombinant topoisomerase iv genes could be attributed to a fitness cost imposed by the horizontal transfer, which often implies the acquisition of larger-than-normal pare-parc intergenic regions. a study of the transcription of these genes and of the fitness cost for 24 isogenic cp r strains was performed. six first-level transformants were obtained either with pcr products containing the parc quinol ... | 2008 | 18160515 |
| early steps in the dna base excision/single-strand interruption repair pathway in mammalian cells. | base excision repair (ber) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ros). ber involves 4-5 steps starting with base excision by a dna glycosylase, followed by a common pathway usually involving an ap-endonuclease (ape) to generate 3' oh terminus at the damage site, followed by repai ... | 2008 | 18166975 |
| kinetic pathway of pyrophosphorolysis by a retrotransposon reverse transcriptase. | dna and rna polymerases use a common phosphoryl transfer mechanism for base addition that requires two or three acidic amino acid residues at their active sites. we previously showed, for the reverse transcriptase (rt) encoded by the yeast retrotransposon ty1, that one of the three conserved active site aspartates (d(211)) can be substituted by asparagine and still retain in vitro polymerase activity, although in vivo transposition is lost. transposition is partially restored by second site supp ... | 2008 | 18167548 |
| structures of open (r) and close (t) states of prephenate dehydratase (pdt)--implication of allosteric regulation by l-phenylalanine. | the enzyme prephenate dehydratase (pdt) converts prephenate to phenylpyruvate in l-phenylalanine biosynthesis. pdt is allosterically regulated by l-phe and other amino acids. we report the first crystal structures of pdt from staphylococcus aureus in a relaxed (r) state and pdt from chlorobium tepidum in a tense (t) state. the two enzymes show low sequence identity (27.3%) but the same prototypic architecture and domain organization. both enzymes are tetramers (dimer of dimers) in crystal and so ... | 2008 | 18171624 |
| structures of open (r) and close (t) states of prephenate dehydratase (pdt)--implication of allosteric regulation by l-phenylalanine. | the enzyme prephenate dehydratase (pdt) converts prephenate to phenylpyruvate in l-phenylalanine biosynthesis. pdt is allosterically regulated by l-phe and other amino acids. we report the first crystal structures of pdt from staphylococcus aureus in a relaxed (r) state and pdt from chlorobium tepidum in a tense (t) state. the two enzymes show low sequence identity (27.3%) but the same prototypic architecture and domain organization. both enzymes are tetramers (dimer of dimers) in crystal and so ... | 2008 | 18171624 |
| identification and role of functionally important motifs in the 970 loop of escherichia coli 16s ribosomal rna. | the 970 loop (helix 31) of escherichia coli 16s ribosomal rna contains two modified nucleotides, m(2)g966 and m(5)c967. positions a964, a969, and c970 are conserved among the bacteria, archaea, and eukarya. the nucleotides present at positions 965, 966, 967, 968, and 971, however, are only conserved and unique within each domain. all organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. biochemical and structure studies have placed this loo ... | 2008 | 18177894 |
| identification and role of functionally important motifs in the 970 loop of escherichia coli 16s ribosomal rna. | the 970 loop (helix 31) of escherichia coli 16s ribosomal rna contains two modified nucleotides, m(2)g966 and m(5)c967. positions a964, a969, and c970 are conserved among the bacteria, archaea, and eukarya. the nucleotides present at positions 965, 966, 967, 968, and 971, however, are only conserved and unique within each domain. all organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. biochemical and structure studies have placed this loo ... | 2008 | 18177894 |
| structural biophysics of the nusb:nuse antitermination complex. | in prokaryotic transcription regulation, several host factors form a complex with rna polymerase and the nascent mrna. as part of a process known as antitermination, two of these host factors, nusb and nuse, bind to form a heterodimer, which interacts with a specific boxa site on the rna. the nusb/nuse/boxa rna ternary complex interacts with the rna polymerase transcription complex, stabilizing it and allowing transcription past premature termination points. the nusb protein also binds boxa rna ... | 2008 | 18177898 |
| structural biophysics of the nusb:nuse antitermination complex. | in prokaryotic transcription regulation, several host factors form a complex with rna polymerase and the nascent mrna. as part of a process known as antitermination, two of these host factors, nusb and nuse, bind to form a heterodimer, which interacts with a specific boxa site on the rna. the nusb/nuse/boxa rna ternary complex interacts with the rna polymerase transcription complex, stabilizing it and allowing transcription past premature termination points. the nusb protein also binds boxa rna ... | 2008 | 18177898 |
| structural insight into the mechanism of substrate specificity of aedes kynurenine aminotransferase. | aedes aegypti kynurenine aminotransferase (aekat) is a multifunctional aminotransferase. it catalyzes the transamination of a number of amino acids and uses many biologically relevant alpha-keto acids as amino group acceptors. aekat also is a cysteine s-conjugate beta-lyase. the most important function of aekat is the biosynthesis of kynurenic acid, a natural antagonist of nmda and alpha7-nicotinic acetylcholine receptors. here, we report the crystal structures of aekat in complex with its best ... | 2008 | 18186649 |
| cross-crystal averaging reveals that the structure of the peptidyl-transferase center is the same in the 70s ribosome and the 50s subunit. | recently, two crystal structures of the thermus thermophilus 70s ribosome in the same functional state were determined at 2.8 and 3.7 a resolution but were different throughout. the most functionally significant structural differences are in the conformation of the peptidyl-transferase center (ptc) and the interface between the ptc and the cca end of the p-site trna. likewise, the 3.7 a ptc differed from the functionally equivalent structure of the haloarcula marismortui 50s subunit. to ascertai ... | 2008 | 18187576 |
| evolution of mal abc transporter operons in the thermococcales and thermotogales. | the mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea thermococcus litoralis and pyrococcus furiosus. bacterial hyperthermophiles of the order thermotogales live among these archaea and so may have shared in these transfers. the genome sequence of thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. we examined deep phylogenetic relationships among the m ... | 2008 | 18197971 |
| nondecarboxylating and decarboxylating isocitrate dehydrogenases: oxalosuccinate reductase as an ancestral form of isocitrate dehydrogenase. | isocitrate dehydrogenase (icdh) from hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. in this study, the oxidation reaction catalyzed by h. thermophilus icdh was kinetically analyzed. as a result, a rapid equilibrium random-order mechanism was suggested. the affinities of both substrates (isocitrate and nad+) toward the enzyme were extremely low ... | 2008 | 18203822 |
| solution structure of ribosomal protein l40e, a unique c4 zinc finger protein encoded by archaeon sulfolobus solfataricus. | the ribosomal protein l40e from archaeon sulfolobus solfataricus is a component of the 50s ribosomal subunit. l40e is a 56-residue, highly basic protein that contains a c4 zinc finger motif, crkc_x(10)_crrc. homologs are found in both archaea and eukaryotes but are not present in bacteria. eukaryotic genomes encode l40e as a ubiquitin-fusion protein. l40e was absent from the crystal structure of euryarchaeota 50s ribosomal subunit. here we report the three-dimensional solution structure of l40e ... | 2008 | 18218710 |
| the solution structure of ribosomal protein s17e from methanobacterium thermoautotrophicum: a structural homolog of the ff domain. | the ribosomal protein s17e from the archaeon methanobacterium thermoautotrophicum is a component of the 30s ribosomal subunit. s17e is a 62-residue protein conserved in archaea and eukaryotes and has no counterparts in bacteria. mammalian s17e is a phosphoprotein component of eukaryotic ribosomes. archaeal s17e proteins range from 59 to 79 amino acids, and are about half the length of the eukaryotic homologs which have an additional c-terminal region. here we report the three-dimensional solutio ... | 2008 | 18218711 |
| molecular and physiological role of the trehalose-hydrolyzing alpha-glucosidase from thermus thermophilus hb27. | trehalose supports the growth of thermus thermophilus strain hb27, but the absence of obvious genes for the hydrolysis of this disaccharide in the genome led us to search for enzymes for such a purpose. we expressed a putative alpha-glucosidase gene (ttc0107), characterized the recombinant enzyme, and found that the preferred substrate was alpha,alpha-1,1-trehalose, a new feature among alpha-glucosidases. the enzyme could also hydrolyze the disaccharides kojibiose and sucrose (alpha-1,2 linkage) ... | 2008 | 18223075 |
| novel monofunctional histidinol-phosphate phosphatase of the dddd superfamily of phosphohydrolases. | the ton_0887 gene was identified as the missing histidinol-phosphate phosphatase (holpase) in the hyperthermophilic archaeon "thermococcus onnurineus" na1. the protein contained conserved motifs of the dddd superfamily of phosphohydrolase, and the recombinantly expressed protein exhibited strong holpase activity. in this study, we functionally assessed for the first time the monofunctional dddd-type holpase, which is organized in the gene cluster. | 2008 | 18223080 |
| novel members of glycoside hydrolase family 13 derived from environmental dna. | starch and pullulan-modifying enzymes of the alpha-amylase family (glycoside hydrolase family 13) have several industrial applications. to date, most of these enzymes have been derived from isolated organisms. to increase the number of members of this enzyme family, in particular of the thermophilic representatives, we have applied a consensus primer-based approach using dna from enrichments from geothermal habitats. with this approach, we succeeded in isolating three new enzymes: a neopullulana ... | 2008 | 18223106 |
| evidence for the involvement of acid/base chemistry in the reaction catalyzed by the type ii isopentenyl diphosphate/dimethylallyl diphosphate isomerase from staphylococcus aureus. | the type ii isopentenyl diphosphate/dimethylallyl diphosphate isomerase (idi-2) is a flavin mononucleotide (fmn)-dependent enzyme that catalyzes the reversible isomerization of isopentenyl pyrophosphate (ipp) to dimethylallyl pyrophosphate (dmapp), a reaction with no net change in redox state of the coenzyme or substrate. here, uv-vis spectral analysis of the idi-2 reaction revealed the accumulation of a reduced neutral dihydroflavin intermediate when the reduced enzyme was incubated with ipp or ... | 2008 | 18229948 |
| structural insights into ribosome recycling factor interactions with the 70s ribosome. | at the end of translation in bacteria, ribosome recycling factor (rrf) is used together with elongation factor g to recycle the 30s and 50s ribosomal subunits for the next round of translation. in x-ray crystal structures of rrf with the escherichia coli 70s ribosome, rrf binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. upon binding of either e. coli or thermus thermophilus rrf to the e. coli ribosome, the tip of ribosomal rna helix 69 in the large ... | 2008 | 18234219 |
| the native cyclobutane pyrimidine dimer photolyase of rice is phosphorylated. | the cyclobutane pyrimidine dimer (cpd) is a major type of dna damage induced by ultraviolet b (uvb) radiation. cpd photolyase, which absorbs blue/uva light as an energy source to monomerize dimers, is a crucial factor for determining the sensitivity of rice (oryza sativa) to uvb radiation. here, we purified native class ii cpd photolyase from rice leaves. as the final purification step, cpd photolyase was bound to cpd-containing dna conjugated to magnetic beads and then released by blue-light ir ... | 2008 | 18235036 |
| functional role of the additional domains in inulosucrase (isla) from leuconostoc citreum cw28. | inulosucrase (isla) from leuconostoc citreum cw28 belongs to a new subfamily of multidomain fructosyltransferases (ftfs), containing additional domains from glucosyltransferases. it is not known what the function of the additional domains in this subfamily is. | 2008 | 18237396 |
| a single mammalian mitochondrial translation initiation factor functionally replaces two bacterial factors. | the mechanism of translation in eubacteria and organelles is thought to be similar. in eubacteria, the three initiation factors if1, if2, and if3 are vital. although the homologs of if2 and if3 are found in mammalian mitochondria, an if1 homolog has never been detected. here, we show that bovine mitochondrial if2 (if2(mt)) complements e. coli containing a deletion of the if2 gene (e. coli deltainfb). we find that if1 is no longer essential in an if2(mt)-supported e. coli deltainfb strain. furthe ... | 2008 | 18243113 |
| cooperative damage recognition by uvra and uvrb: identification of uvra residues that mediate dna binding. | nucleotide excision repair (ner) is responsible for the recognition and removal of numerous structurally unrelated dna lesions. in prokaryotes, the proteins uvra, uvrb and uvrc orchestrate the recognition and excision of aberrant lesions from dna. despite the progress we have made in understanding the ner pathway, it remains unclear how the uvra dimer interacts with dna to facilitate dna damage recognition. the purpose of this study was to define amino acid residues in uvra that provide binding ... | 2008 | 18248777 |
| functional interaction between bases c1049 in domain ii and g2751 in domain vi of 23s rrna in escherichia coli ribosomes. | the factor-binding center within the escherichia coli ribosome is comprised of two discrete domains of 23s rrna: the gtpase-associated region (gar) in domain ii and the sarcin-ricin loop in domain vi. these two regions appear to collaborate in the factor-dependent events that occur during protein synthesis. current x-ray crystallography of the ribosome shows an interaction between c1049 in the gar and g2751 in domain vi. we have confirmed this interaction by site-directed mutagenesis and chemica ... | 2008 | 18252772 |
| rimo, a miab-like enzyme, methylthiolates the universally conserved asp88 residue of ribosomal protein s12 in escherichia coli. | ribosomal protein s12 undergoes a unique posttranslational modification, methylthiolation of residue d88, in escherichia coli and several other bacteria. using mass spectrometry, we have identified the enzyme responsible for this modification in e. coli, the ylig gene product. this enzyme, which we propose be called rimo, is a radical-s-adenosylmethionine protein that bears strong sequence similarity to miab, which methylthiolates trna. we show that rimo and miab represent two of four subgroups ... | 2008 | 18252828 |
| functional importance of individual rrna 2'-o-ribose methylations revealed by high-resolution phenotyping. | ribosomal rnas contain numerous modifications at specific nucleotides. despite their evolutionary conservation, the functional role of individual 2'-o-ribose methylations in rrna is not known. a distinct family of small nucleolar rnas, box c/d snornas, guides the methylating complex to specific rrna sites. using a high-resolution phenotyping approach, we characterized 20 box c/d snorna gene deletions for altered growth dynamics under a wide array of environmental perturbations, encompassing intr ... | 2008 | 18256246 |
| crystallization and preliminary x-ray characterization of qued from bacillus subtilis, an enzyme involved in queuosine biosynthesis. | qued (previously named ykvk) is one of several enzymes involved in the biosynthesis of the hypermodified nucleoside queuosine. queuosine is incorporated into trna at position 34 of four trnas: trna(his), trna(asp), trna(asn) and trna(tyr). the crystallization and preliminary x-ray crystallographic studies of qued are described here. the recombinant protein from bacillus subtilis was overproduced in escherichia coli and crystallized using the hanging-drop vapor-diffusion method from 25% peg 600, ... | 2008 | 18259064 |
| structure/function analysis of yeast ribosomal protein l2. | ribosomal protein l2 is a core element of the large subunit that is highly conserved among all three kingdoms. l2 contacts almost every domain of the large subunit rrna and participates in an intersubunit bridge with the small subunit rrna. it contains a solvent-accessible globular domain that interfaces with the solvent accessible side of the large subunit that is linked through a bridge to an extension domain that approaches the peptidyltransferase center. here, screening of randomly generated ... | 2008 | 18263608 |
| a novel chromate reductase from thermus scotoductus sa-01 related to old yellow enzyme. | bacteria can reduce toxic and carcinogenic cr(vi) to insoluble and less toxic cr(iii). thermus scotoductus sa-01, a south african gold mine isolate, has been shown to be able to reduce a variety of metals, including cr(vi). here we report the purification to homogeneity and characterization of a novel chromate reductase. the oxidoreductase is a homodimeric protein, with a monomer molecular mass of approximately 36 kda, containing a noncovalently bound flavin mononucleotide cofactor. the chromate ... | 2008 | 18263719 |
| decreased aminoacylation in pathology-related mutants of mitochondrial trnatyr is associated with structural perturbations in trna architecture. | a growing number of human pathologies are ascribed to mutations in mitochondrial trna genes. here, we report biochemical investigations on three mt-trna(tyr) molecules with point substitutions associated with diseases. the mutations occur in the atypical t- and d-loops at positions homologous to those involved in the tertiary interaction network of canonical trnas. they do not correspond to tyrosine identity positions and likely do not contact the mitochondrial tyrosyl-trna synthetase during the ... | 2008 | 18268021 |
| ph-dependent structural changes of helix 69 from escherichia coli 23s ribosomal rna. | helix 69 in 23s rrna is a region in the ribosome that participates in a considerable number of rna-rna and rna-protein interactions. conformational flexibility is essential for such a region to interact and accommodate protein factors at different stages of protein biosynthesis. in this study, ph-dependent structural and stability changes were observed for helix 69 through a variety of spectroscopic techniques, such as circular dichroism spectroscopy, uv melting, and nuclear magnetic resonance s ... | 2008 | 18268024 |
| genetic and chemical modifiers of a cug toxicity model in drosophila. | non-coding cug repeat expansions interfere with the activity of human muscleblind-like (mbnl) proteins contributing to myotonic dystrophy 1 (dm1). to understand this toxic rna gain-of-function mechanism we developed a drosophila model expressing 60 pure and 480 interrupted cug repeats in the context of a non-translatable rna. these flies reproduced aspects of the dm1 pathology, most notably nuclear accumulation of cug transcripts, muscle degeneration, splicing misregulation, and diminished muscl ... | 2008 | 18270582 |
| still looking for the magic spot: the crystallographically defined binding site for ppgpp on rna polymerase is unlikely to be responsible for rrna transcription regulation. | identification of the rna polymerase (rnap) binding site for ppgpp, a central regulator of bacterial transcription, is crucial for understanding its mechanism of action. a recent high-resolution x-ray structure defined a ppgpp binding site on thermus thermophilus rnap. we report here effects of ppgpp on 10 mutant escherichia coli rnaps with substitutions for the analogous residues within 3-4 a of the ppgpp binding site in the t. thermophilus cocrystal. none of the substitutions in e. coli rnap s ... | 2008 | 18272182 |
| expression of recombinant rhipicephalus (boophilus) microplus, r. annulatus and r. decoloratus bm86 orthologs as secreted proteins in pichia pastoris. | rhipicephalus (boophilus) spp. ticks economically impact on cattle production in africa and other tropical and subtropical regions of the world. tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. the r. microplus bm86 protective antigen has been produced by recombinant dna technology and shown to protect cattle against tick infestations. | 2008 | 18275601 |