| simultaneous detection of measles virus, rubella virus, and parvovirus b19 by using multiplex pcr. | we describe here a multiplex reverse transcription-pcr (rtmnpcr) assay designed to detect and differentiate measles virus, rubella virus, and parvovirus b19. serial dilution experiments with vaccine strains that compared cell culture isolation of measles in b95 cells and rubella in rk13 cells showed sensitivity rates of 0.004 50% tissue culture infective dose (tcid(50)) for measles virus and 0.04 tcid(50) for rubella virus. this rtmnpcr can detect as few as 10 molecules for measles virus and rub ... | 2002 | 11773102 |
| recombinational transfer of 100-kilobase genomic dna to plasmid in bacillus subtilis 168. | transformation of bacillus subtilis by a plasmid requires a circular multimeric form. in contrast, linearized plasmids can be circularized only when homologous sequences are present in the host genome. a recombinational transfer system was constructed with this intrinsic b. subtilis recombinational repair pathway. the vector, pgets103, a derivative of the theta-type replicating plasmid ptb19 of thermophilic bacillus, had the full length of escherichia coli plasmid pbr322. a multimeric form of pg ... | 2001 | 11514534 |
| a conformational change in the ribosomal peptidyl transferase center upon active/inactive transition. | the ribosome is a dynamic particle that undergoes many structural changes during translation. we show through chemical probing with dimethyl sulfate (dms) that conformational changes occur at several nucleotides in the peptidyl transferase center upon alterations in ph, temperature, and monovalent ion concentration, consistent with observations made by elson and coworkers over 30 years ago. moreover, we have found that the ph-dependent dms reactivity of a2451 in the center of the 23s rrna peptid ... | 2001 | 11517305 |
| contributions of folding cores to the thermostabilities of two ribonucleases h. | to investigate the contribution of the folding cores to the thermodynamic stability of rnases h, we used rational design to create two chimeras composed of parts of a thermophilic and a mesophilic rnase h. each chimera combines the folding core from one parent protein and the remaining parts of the other. both chimeras form active, well-folded rnases h. stability curves, based on cd-monitored chemical denaturations, show that the chimera with the thermophilic core is more stable, has a higher mi ... | 2002 | 11790848 |
| the structure of an asprs-trna(asp) complex reveals a trna-dependent control mechanism. | the 2.6 a resolution crystal structure of an inactive complex between yeast trna(asp) and escherichia coli aspartyl-trna synthetase reveals the molecular details of a trna-induced mechanism that controls the specificity of the reaction. the dimer is asymmetric, with only one of the two bound trnas entering the active site cleft of its subunit. however, the flipping loop, which controls the proper positioning of the amino acid substrate, acts as a lid and prevents the correct positioning of the t ... | 2001 | 11566892 |
| tetracycline induces stabilization of mrna in bacillus subtilis. | the tet(l) gene of bacillus subtilis confers low-level tetracycline (tc) resistance. previous work examining the >20-fold-inducible expression of tet(l) by tc demonstrated a 12-fold translational induction. here we show that the other component of tet(l) induction is at the level of mrna stabilization. addition of a subinhibitory concentration of tc results in a two- to threefold increase in tet(l) mrna stability. using a plasmid-borne derivative of tet(l) with a large in-frame deletion of the c ... | 2002 | 11807047 |
| analysis of the levels of conservation of the j domain among the various types of dnaj-like proteins. | dnaj-like proteins are defined by the presence of an approximately 73 amino acid region termed the j domain. this region bears similarity to the initial 73 amino acids of the escherichia coli protein dnaj. although the structures of the j domains of e coli dnaj and human heat shock protein 40 have been solved using nuclear magnetic resonance, no detailed analysis of the amino acid conservation among the j domains of the various dnaj-like proteins has yet been attempted. a multiple alignment of 2 ... | 2000 | 11048657 |
| multiple lateral transfers of dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes. | a large fragment of the dissimilatory sulfite reductase genes (dsrab) was pcr amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. in addition, the sequence of the dsrab gene homologs of the sulfite reducer desulfitobacterium dehalogenans was determined. in contrast to previous reports, comparative analysis of all available dsrab sequences produced a tree topology partially inconsistent with the corresponding 16s rrna phylogen ... | 2001 | 11567003 |
| trna aminoacylation by arginyl-trna synthetase: induced conformations during substrates binding. | the 2.2 a crystal structure of a ternary complex formed by yeast arginyl-trna synthetase and its cognate trna(arg) in the presence of the l-arginine substrate highlights new atomic features used for specific substrate recognition. this first example of an active complex formed by a class ia aminoacyl-trna synthetase and its natural cognate trna illustrates additional strategies used for specific trna selection. the enzyme specifically recognizes the d-loop and the anticodon of the trna, and the ... | 2000 | 11060012 |
| molecular analyses of the natural transformation machinery and identification of pilus structures in the extremely thermophilic bacterium thermus thermophilus strain hb27. | thermus thermophilus hb27, an extremely thermophilic bacterium, exhibits high competence for natural transformation. to identify genes of the natural transformation machinery of t. thermophilus hb27, we performed homology searches in the partially completed t. thermophilus genomic sequence for conserved competence genes. these analyses resulted in the detection of 28 open reading frames (orfs) exhibiting significant similarities to known competence proteins of gram-negative and gram-positive bac ... | 2002 | 11823215 |
| corynebacterium glutamicum utilizes both transsulfuration and direct sulfhydrylation pathways for methionine biosynthesis. | a direct sulfhydrylation pathway for methionine biosynthesis in corynebacterium glutamicum was found. the pathway was catalyzed by mety encoding o-acetylhomoserine sulfhydrylase. the gene mety, located immediately upstream of meta, was found to encode a protein of 437 amino acids with a deduced molecular mass of 46,751 da. in accordance with dna and protein sequence data, the introduction of mety into c. glutamicum resulted in the accumulation of a 47-kda protein in the cells and a 30-fold incre ... | 2002 | 11844756 |
| structural analysis of an escherichia coli endonuclease viii covalent reaction intermediate. | endonuclease viii (nei) of escherichia coli is a dna repair enzyme that excises oxidized pyrimidines from dna. nei shares with formamidopyrimidine-dna glycosylase (fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential beta-elimination steps. however, nei differs significantly from fpg in substrate specificity. we determined the structure of nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 a re ... | 2002 | 11847126 |
| identification of a mycobacterium tuberculosis putative classical nitroreductase gene whose expression is coregulated with that of the acr aene within macrophages, in standing versus shaking cultures, and under low oxygen conditions. | tuberculosis remains a leading killer worldwide, and new approaches for its treatment and prevention are urgently needed. this effort will benefit greatly from a better understanding of gene regulation in mycobacterium tuberculosis, particularly with respect to this pathogen's response to its host environment. we examined the behavior of two promoters from the divergently transcribed m. tuberculosis genes acr/hspx/rv2031c (alpha-crystallin homolog) and rv2032/acg (acr-coregulated gene) by using ... | 2002 | 11854240 |
| trna-like recognition of group i introns by a tyrosyl-trna synthetase. | the neurospora crassa mitochondrial tyrosyl-trna synthetase (cyt-18 protein) functions in splicing group i introns by promoting the formation of the catalytically active rna structure. previous work suggested that cyt-18 recognizes a conserved trna-like structure of the group i intron catalytic core. here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and c-terminal domains of cyt-18 interact with the expected group i intron cognates of the aminoacyl-acceptor st ... | 2002 | 11854463 |
| crystal structure combined with genetic analysis of the thermus thermophilus ribosome recycling factor shows that a flexible hinge may act as a functional switch. | ribosome recycling factor (rrf), in concert with elongation factor ef-g, is required for disassembly of the posttermination complex of the ribosome after release of polypeptides. the crystal structure of thermus thermophilus rrf was determined at 2.6 a resolution. it is a trna-like l-shaped molecule consisting of two domains: a long three-helix bundle (domain 1) and a three-layer beta/alpha/beta sandwich (domain 2). although the individual domain structures are similar to those of thermotoga mar ... | 2000 | 11073219 |
| identification and comparative analysis of the chloroplast alpha-subunit gene of dna-dependent rna polymerase from seven euglena species. | when the sequence of the euglena gracilis chloroplast genome was reported in 1993 the alpha-subunit gene (rpoa) of rna polymerase appeared to be missing, based on a comparison of all putative reading frames to the then known rpoa loci. since there has been a large increase in known rpoa sequences, the question of a euglena chloroplast rpoa gene was re-examined. a previously described unknown reading frame of 161 codons was found to be part of an rpoa gene split by a single group iii intron. this ... | 2002 | 11861918 |
| characterization of rnase p from thermotoga maritima. | the protein subunit of rnase p from a thermophilic bacterium, thermotoga maritima, was overexpressed in and purified from escherichia coli. the cloned protein was reconstituted with the rna subunit transcribed in vitro. the temperature optimum of the holoenzyme is near 50 degrees c, with no enzymatic activity at 65 degrees c or above. this finding is in sharp contrast to the optimal growth temperature of t.maritima, which is near 80 degrees c. however, in heterologous reconstitution experiments ... | 2001 | 11160919 |
| transfer rna-dependent amino acid biosynthesis: an essential route to asparagine formation. | biochemical experiments and genomic sequence analysis showed that deinococcus radiodurans and thermus thermophilus do not possess asparagine synthetase (encoded by asna or asnb), the enzyme forming asparagine from aspartate. instead these organisms derive asparagine from asparaginyl-trna, which is made from aspartate in the trna-dependent transamidation pathway [becker, h. d. & kern, d. (1998) proc. natl. acad. sci. usa 95, 12832-12837; and curnow, a. w., tumbula, d. l., pelaschier, j. t., min, ... | 2002 | 11880622 |
| filamentous phage active on the gram-positive bacterium propionibacterium freudenreichii. | we present the first description of a single-stranded dna filamentous phage able to replicate in a gram-positive bacterium. phage b5 infects propionibacterium freudenreichii and has a genome consisting of 5,806 bases coding for 10 putative open reading frames. the organization of the genome is very similar to the organization of the genomes of filamentous phages active on gram-negative bacteria. the putative coat protein exhibits homology with the coat proteins of phages ph75 and pf3 active on t ... | 2002 | 11889111 |
| prediction of structural domains of tap reveals details of its interaction with p15 and nucleoporins. | vertebrate tap is a nuclear mrna export factor homologous to yeast mex67p. the middle domain of tap binds directly to p15, a protein related to the nuclear transport factor 2 (ntf2), whereas its c-terminal domain interacts with various nucleoporins, the components of the nuclear pore complex (npc). here, we report that the middle domain of tap is also similar to ntf2, as well as to regions in ras-gap sh3 domain binding protein (g3bp) and some plant protein kinases. based on the known three-dimen ... | 2000 | 11256625 |
| mutations in the 16s rrna genes of helicobacter pylori mediate resistance to tetracycline. | low-cost and rescue treatments for helicobacter pylori infections involve combinations of several drugs including tetracycline. resistance to tetracycline has recently emerged in h. pylori. the 16s rrna gene sequences of two tetracycline-resistant clinical isolates (mic = 64 microg/ml) were determined and compared to the consensus h. pylori 16s rrna sequence. one isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. natural transformation with the 16s ... | 2002 | 11914344 |
| reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing saccharomyces cerevisiae strains improves the ethanol yield from xylose. | in recombinant, xylose-fermenting saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. xylitol production results from a cofactor imbalance, since xylose reductase uses both nadph and nadh, while xylitol dehydrogenase uses only nad(+). in this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the nadph-producing pentose phosphate pathway. the pentose phosphate pathway was blocked either by disruption of the gnd1 gene, ... | 2002 | 11916674 |
| use of fe(iii) as an electron acceptor to recover previously uncultured hyperthermophiles: isolation and characterization of geothermobacterium ferrireducens gen. nov., sp. nov. | it has recently been recognized that the ability to use fe(iii) as a terminal electron acceptor is a highly conserved characteristic in hyperthermophilic microorganisms. this suggests that it may be possible to recover as-yet-uncultured hyperthermophiles in pure culture if fe(iii) is used as an electron acceptor. as part of a study of the microbial diversity of the obsidian pool area in yellowstone national park, wyo., hot sediment samples were used as the inoculum for enrichment cultures in med ... | 2002 | 11916691 |
| efficient trans-cleavage by the schistosoma mansoni smalpha1 hammerhead ribozyme in the extreme thermophile thermus thermophilus. | the catalytic hammerhead structure has been found in association with repetitive dna from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer rna in vivo. the cellular role of these repetitive elements and their transcripts is unknown. moreover, none of these natural hammerheads have been shown to trans-cleave a host mrna in vivo. we analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme ... | 2002 | 11917021 |
| tspgwi, a thermophilic class-iis restriction endonuclease from thermus sp., recognizes novel asymmetric sequence 5'-acgga(n11/9)-3'. | a novel prototype class-iis restriction endonuclease, tspgwi, was isolated from the thermophilic bacterium thermus sp. gw. the recognition sequence and cleavage positions have been established: tspgwi recognizes the non-palindromic 5-bp sequence 5'-acgga-3' and cleaves the dna 11 and 9 nt downstream in the top and bottom strand, respectively. in addition, an accompanying endonuclease, tspgwii, an isoschizomer of pst i, was found in thermus sp. gw cells. | 2002 | 11917039 |
| peptide deformylase as an antibacterial drug target: assays for detection of its inhibition in escherichia coli cell homogenates and intact cells. | an assay was developed to determine the activity of peptide deformylase (pdf) inhibitors under conditions as close as possible to the physiological situation. the assay principle is the detection of n-terminal [35s]methionine labeling of a protein that contains no internal methionine. if pdf is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the n-terminal methionine label. in the presence of a pdf inhibitor, ... | 2001 | 11257015 |
| peptide deformylase as an antibacterial drug target: target validation and resistance development. | new inhibitors of peptide deformylase (pdf) which are very potent against the isolated enzyme and show a certain degree of antibacterial activity have recently been synthesized by our group. several lines of experimental evidence indicate that these inhibitors indeed interfere with the target enzyme in the bacterial cell. (i) the inhibition of escherichia coli growth could be counteracted by overexpression of pdf from different organisms, including e. coli, streptococcus pneumoniae, and haemophi ... | 2001 | 11257016 |
| stability and interactions of the amino-terminal domain of clpb from escherichia coli. | clpb is a member of a multichaperone system in escherichia coli (with dnak, dnaj, and grpe) that reactivates aggregated proteins. the sequence of clpb contains two atp-binding regions that are enclosed between the n- and c-terminal extensions. whereas it has been found that the n-terminal region of clpb is essential for the chaperone activity, the structure of this region is not known, and its biochemical properties have not been studied. we expressed and purified the n-terminal fragment of clpb ... | 2002 | 11967375 |
| purification and characterization of the recombinant thermus sp. strain t2 alpha-galactosidase expressed in escherichia coli. | the nucleotide sequence of the thermus sp. strain t2 dna coding for a thermostable alpha-galactosidase was determined. the deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (m(r), 53,514). the observed homology between the deduced amino acid sequences of the enzyme and alpha-galactosidase from thermus brockianus was over 70%. thermus sp. strain t2 alpha-galactosidase was expressed in its active form in escherichia coli and purified. native polyacrylamide gel ele ... | 2001 | 11282611 |
| importance of the conserved nucleotides around the trna-like structure of escherichia coli transfer-messenger rna for protein tagging. | a bacterial rna functioning as both trna and mrna, transfer-messenger rna (tmrna) rescues stalled ribosomes and clears the cell of incomplete polypeptides. for function, escherichia coli tmrna requires an elaborate interplay between a trna-like structure and an internal mrna domain that are connected by a 295 nt long compact secondary structure. the trna-like structure is surrounded by 16 unpaired nt, including 10 residues that are >95% conserved among the known 140 tmrna sequences. all these re ... | 2001 | 11713316 |
| infection of tick cells and bovine erythrocytes with one genotype of the intracellular ehrlichia anaplasma marginale excludes infection with other genotypes. | anaplasma marginale, a tick-borne rickettsial pathogen of cattle, is endemic in several areas of the united states. many geographic isolates of a. marginale that occur in the united states are characterized by the major surface protein 1a, which varies in sequence and molecular weight due to different numbers of tandem repeats of 28 or 29 amino acids. recent studies (g. h. palmer, f. r. rurangirwa, and t. f. mcelwain, j. clin. microbiol. 39:631-635, 2001) of an a. marginale-infected herd of catt ... | 2002 | 11986275 |
| protein coding palindromes are a unique but recurrent feature in rickettsia. | rickettsia are unique in inserting in-frame a number of palindromic sequences within protein coding regions. in this study, we extensively analyzed repeated sequences in the genome of rickettsia conorii and examined their locations in regard to coding versus noncoding regions. we identified 656 interspersed repeated sequences classified into 10 distinct families. of the 10 families, three palindromic sequence families showed clear cases of insertions into open reading frames (orfs). the location ... | 2002 | 11997347 |
| adp-dependent phosphofructokinases in mesophilic and thermophilic methanogenic archaea. | phosphofructokinase (pfk) is a key enzyme of the glycolytic pathway in all domains of life. two related pfks, atp-dependent and pp(i)-dependent pfk, have been distinguished in bacteria and eucarya, as well as in some archaea. hyperthermophilic archaea of the order thermococcales, including pyrococcus and thermococcus spp., have recently been demonstrated to possess a unique adp-dependent pfk (adp-pfk) that appears to be phylogenetically distinct. here, we report the presence of adp-pfks in glyco ... | 2001 | 11717273 |
| occurrence of leu+ revertants under starvation cultures in escherichia coli is growth-dependent. | many investigations have reported that advantageous mutations occurred more frequently under selective conditions than those under non-selective conditions. this phenomenon is referred to as adaptive mutation. their characteristics are that adaptive mutations are directed and growth-independent. the idea of directed adaptive mutation had been objected by some reports, however, the idea of growth-independent adaptive mutation has been held till today. | 2002 | 12019019 |
| v-shaped structure of glutamyl-trna reductase, the first enzyme of trna-dependent tetrapyrrole biosynthesis. | processes vital to life such as respiration and photosynthesis critically depend on the availability of tetrapyrroles including hemes and chlorophylls. trna-dependent catalysis generally is associated with protein biosynthesis. an exception is the reduction of glutamyl-trna to glutamate-1-semialdehyde by the enzyme glutamyl-trna reductase. this reaction is the indispensable initiating step of tetrapyrrole biosynthesis in plants and most prokaryotes. the crystal structure of glutamyl-trna reducta ... | 2001 | 11726494 |
| complete polar lipid composition of thermoplasma acidophilum ho-62 determined by high-performance liquid chromatography with evaporative light-scattering detection. | polar ether lipids of thermoplasma acidophilum ho-62 were purified by high-performance liquid chromatography with an evaporative light-scattering detector. structures of purified lipids were investigated by capillary gas chromatography, mass spectrometry, and nuclear magnetic resonance. three types of ether lipids were found: phospholipids, glycolipids, and phosphoglycolipids. the two phospholipids had glycerophosphate as the phosphoester moiety. the seven glycolipids had different combinations ... | 2002 | 11751835 |
| cloning, sequencing, and expression of the cold-inducible hutu gene from the antarctic psychrotrophic bacterium pseudomonas syringae. | a promoter-fusion study with a tn 5-based promoter probe vector had earlier found that the hutu gene which encodes the enzyme urocanase for the histidine utilization pathway is upregulated at a lower temperature (4 degrees c) in the antarctic psychrotrophic bacterium pseudomonas syringae. to examine the characteristics of the urocanase gene and its promoter elements from the psychrotroph, the complete hutu and its upstream region from p. syringae were cloned, sequenced, and analyzed in the prese ... | 2002 | 11772602 |
| cooperative kinetics of both hsp104 atpase domains and interdomain communication revealed by aaa sensor-1 mutants. | aaa proteins share a conserved active site for atp hydrolysis and regulate many cellular processes. aaa proteins are oligomeric and often have multiple atpase domains per monomer, which is suggestive of complex allosteric kinetics of atp hydrolysis. here, using wild-type hsp104 in the hexameric state, we demonstrate that its two aaa modules (nbd1 and nbd2) have very different catalytic activities, but each displays cooperative kinetics of hydrolysis. using mutations in the aaa sensor-1 motif of ... | 2002 | 11782421 |
| blocking site-to-site translocation of a misactivated amino acid by mutation of a class i trna synthetase. | the genetic code is established by the aminoacylation reactions of trna synthetases. its accuracy depends on editing reactions that prevent amino acids from being assigned to incorrect codons. a group of class i synthetases share a common insertion that encodes a distinct site for editing that is about 30 a from the active site. both misactivated aminoacyl adenylates and mischarged amino acids attached to trna are translocated to this site, which, in turn, is divided into subsites--one for the a ... | 2002 | 11782529 |
| cell-surface-anchoring role of n-terminal surface layer homology domains of clostridium cellulovorans enge. | enge, coding for endoglucanase e, one of the three major subunits of the clostridium cellulovorans cellulosome, has been cloned and sequenced (y. tamaru and r. h. doi, j. bacteriol. 181:3270-3276, 1999). the n-terminal-half region of enge possesses three repeated surface layer homology (slh) domains, which are homologous to those of some bacterial s-layer proteins. also, the c-terminal-half region consists of a catalytic domain of glycosyl hydrolase family 5 and a duplicated sequence (dockerin) ... | 2002 | 11807046 |
| a membrane-bound archaeal lon protease displays atp-independent proteolytic activity towards unfolded proteins and atp-dependent activity for folded proteins. | in contrast to the eucaryal 26s proteasome and the bacterial atp-dependent proteases, little is known about the energy-dependent proteolysis in members of the third domain, archae. we cloned a gene homologous to atp-dependent lon protease from a hyperthermophilic archaeon and observed the unique properties of the archaeal lon. lon from thermococcus kodakaraensis kod1 (lon(tk)) is a 70-kda protein with an n-terminal atpase domain belonging to the aaa(+) superfamily and a c-terminal protease domai ... | 2002 | 12057965 |
| crystal structure of the lactococcus lactis formamidopyrimidine-dna glycosylase bound to an abasic site analogue-containing dna. | the formamidopyrimidine-dna glycosylase (fpg, mutm) is a bifunctional base excision repair enzyme (dna glycosylase/ap lyase) that removes a wide range of oxidized purines, such as 8-oxoguanine and imidazole ring-opened purines, from oxidatively damaged dna. the structure of a non-covalent complex between the lactoccocus lactis fpg and a 1,3-propanediol (pr) abasic site analogue-containing dna has been solved. through an asymmetric interaction along the damaged strand and the intercalation of the ... | 2002 | 12065399 |
| mode of dna-protein interaction between the c-terminal domain of escherichia coli rna polymerase alpha subunit and t7d promoter up element. | the c-terminal domain (ctd) downstream from residue 235 of escherichia coli rna polymerase alpha subunit is involved in recognition of the promoter up element. here we have demonstrated, by dnase i and hydroxyl radical mapping, the presence of two up element subsites on the promoter d of phage t7, each located half and one-and-a-half helix turns, respectively, upstream from the promoter -35 element. this non-typical up element retained its alphactd-binding capability when transferred into the ge ... | 2001 | 11812819 |
| resistance to quinupristin-dalfopristin due to mutation of l22 ribosomal protein in staphylococcus aureus. | the mechanism of resistance to the streptogramin antibiotics quinupristin and dalfopristin was studied in a staphylococcus aureus clinical isolate selected under quinupristin-dalfopristin therapy, in four derivatives of s. aureus rn4220 selected in vitro, and in a mutant selected in a model of rabbit aortic endocarditis. for all strains the mics of erythromycin, quinupristin, and quinupristin-dalfopristin were higher than those for the parental strains but the mics of dalfopristin and lincomycin ... | 2002 | 12069975 |
| immunization of rhesus macaques with a dna prime/modified vaccinia virus ankara boost regimen induces broad simian immunodeficiency virus (siv)-specific t-cell responses and reduces initial viral replication but does not prevent disease progression following challenge with pathogenic sivmac239. | producing a prophylactic vaccine for human immunodeficiency virus (hiv) has proven to be a challenge. most biological isolates of hiv are difficult to neutralize, so that conventional subunit-based antibody-inducing vaccines are unlikely to be very effective. in the rhesus macaque model, some protection was afforded by dna/recombinant viral vector vaccines. however, these studies used as the challenge virus shiv-89.6p, which is neutralizable, making it difficult to determine whether the observed ... | 2002 | 12072518 |
| novel dna-binding proteins in the cyanobacterium anabaena sp. strain pcc 7120. | as an approach towards elucidation of the biochemical regulation of the progression of heterocyst differentiation in anabaena sp. strain pcc 7120, we have identified proteins that bind to a 150-bp sequence upstream from hepc, a gene that plays a role in the synthesis of heterocyst envelope polysaccharide. such proteins were purified in four steps from extracts of vegetative cells of anabaena sp. two of these proteins (abp1 and abp2) are encoded by neighboring genes in the anabaena sp. chromosome ... | 2002 | 12081965 |
| a protonated base pair participating in rrna tertiary structural interactions. | in the recently published x-ray crystallographic structure for the 50s subunit of haloarcula marismortui ribosomes, residue u2546 of the 23s rrna forms a non-watson-crick base pair with u2610. the corresponding residues in the secondary structure of the escherichia coli 23s molecule are u2511 and c2575, and it follows that the latter base (c2575) should be protonated in order to form a base pair that is isostructural with its counterpart in h.marismortui. this prediction was demonstrated experim ... | 2001 | 11812838 |
| nmr structure of a ribosomal rna hairpin containing a conserved cucaa pentaloop. | the structure of a 23 nt rna sequence, rggacccgggcucaaccugggucc, was elucidated using homonuclear nmr, distance geometry and restrained molecular dynamics. this rna is analogous to residues 612-628 of the escherichia coli 16s rrna. the structure of the rna reveals the presence of a pentaloop closed by a duplex stem in typical a-form conformation. the loop does not form a u-turn motif, as previously predicted. a non-planar a.c.a triple base interaction (hydrogen bonds a13 nh6-c10 o2 and c10 n3-a1 ... | 2001 | 11812846 |
| visualization of protein s1 within the 30s ribosomal subunit and its interaction with messenger rna. | s1 is the largest ribosomal protein, present in the small subunit of the bacterial ribosome. it has a pivotal role in stabilizing the mrna on the ribosome. thus far, s1 has eluded structural determination. we have identified the s1 protein mass in the cryo-electron microscopic map of the escherichia coli ribosome by comparing the map with a recent x-ray crystallographic structure of the 30s subunit, which lacks s1. according to our finding, s1 is located at the junction of head, platform, and ma ... | 2001 | 11593008 |
| the unique tuf2 gene from the kirromycin producer streptomyces ramocissimus encodes a minor and kirromycin-sensitive elongation factor tu. | streptomyces ramocissimus, the producer of elongation factor tu (ef-tu)-targeted antibiotic kirromycin, contains three divergent tuf-like genes, with tuf1 encoding regular kirromycin-sensitive ef-tu1; the functions of tuf2 and tuf3 are unknown. analysis of the tuf gene organization in nine producers of kirromycin-type antibiotics revealed that they all contain homologues of tuf1 and sometimes of tuf3 but that tuf2 was found in s. ramocissimus only. the tuf2-flanking regions were sequenced, and t ... | 2002 | 12107139 |
| genomic and functional analyses of sxt, an integrating antibiotic resistance gene transfer element derived from vibrio cholerae. | sxt is representative of a family of conjugative-transposon-like mobile genetic elements that encode multiple antibiotic resistance genes. in recent years, sxt-related conjugative, self-transmissible integrating elements have become widespread in asian vibrio cholerae. we have determined the 100-kb dna sequence of sxt. this element appears to be a chimera composed of transposon-associated antibiotic resistance genes linked to a variety of plasmid- and phage-related genes, as well as to many gene ... | 2002 | 12107144 |
| mutant analysis shows that alanine racemases from pseudomonas aeruginosa and escherichia coli are dimeric. | alanine racemases are ubiquitous prokaryotic enzymes providing the essential peptidoglycan precursor d-alanine. we present evidence that the enzymes from pseudomonas aeruginosa and escherichia coli function exclusively as homodimers. moreover, we demonstrate that expression of a k35a y235a double mutation of dadx in e. coli suppresses bacterial growth in a dominant negative fashion. | 2002 | 12107154 |
| class i tyrosyl-trna synthetase has a class ii mode of cognate trna recognition. | bacterial tyrosyl-trna synthetases (tyrrs) possess a flexibly linked c-terminal domain of approximately 80 residues, which has hitherto been disordered in crystal structures of the enzyme. we have determined the structure of thermus thermophilus tyrrs at 2.0 a resolution in a crystal form in which the c-terminal domain is ordered, and confirm that the fold is similar to part of the c-terminal domain of ribosomal protein s4. we have also determined the structure at 2.9 a resolution of the complex ... | 2002 | 12110594 |
| pcr-based ordered genomic libraries: a new approach to drug target identification for streptococcus pneumoniae. | described here are the development and validation of a novel approach to identify genes encoding drug targets in streptococcus pneumoniae. the method relies on the use of an ordered genomic library composed of pcr amplicons that were generated under error-prone conditions so as to introduce random mutations into the dna. since some of the mutations occur in drug target-encoding genes and subsequently affect the binding of the drug to its respective cellular target, amplicons containing drug targ ... | 2002 | 12121925 |
| regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation. | the riboflavin biosynthesis in bacteria was analyzed using comparative analysis of genes, operons and regulatory elements. a model for regulation based on formation of alternative rna structures involving the rfn elements is suggested. in gram-positive bacteria including actinomycetes, thermotoga, thermus and deinococcus, the riboflavin metabolism and transport genes are predicted to be regulated by transcriptional attenuation, whereas in most gram-negative bacteria, the riboflavin biosynthesis ... | 2002 | 12136096 |
| functional annotation of class i lysyl-trna synthetase phylogeny indicates a limited role for gene transfer. | functional and comparative genomic studies have previously shown that the essential protein lysyl-trna synthetase (lysrs) exists in two unrelated forms. most prokaryotes and all eukaryotes contain a class ii lysrs, whereas most archaea and a few bacteria contain a less common class i lysrs. in bacteria the class i lysrs is only found in the alpha-proteobacteria and a scattering of other groups, including the spirochetes, while the class i protein is by far the most common form of lysrs in archae ... | 2002 | 12142429 |
| biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil. | isolated soil dna from an oak-hornbeam forest close to cologne, germany, was suitable for pcr amplification of gene segments coding for the 16s rrna and nitrogenase reductase (nifh), nitrous oxide reductase (nosz), cytochrome cd(1)-containing nitrite reductase (nirs), and cu-containing nitrite reductase (nirk) of denitrification. for each gene segment, diverse pcr products were characterized by cloning and sequencing. none of the 16s rrna gene sequences was identical to any deposited in the data ... | 2002 | 12147477 |
| experimental evaluation of topological parameters determining protein-folding rates. | recent work suggests that structural topology plays a key role in determining protein-folding rates and pathways. the refolding rates of small proteins that fold without intermediates are found to correlate with simple structural parameters such as relative contact order, long-range order, or the fraction of short-range contacts. to test and evaluate the role of structural topology experimentally, a set of circular permutants of the ribosomal protein s6 from thermus thermophilus was analyzed. de ... | 2002 | 12149462 |
| the methanobacterium thermoautotrophicum mcm protein can form heptameric rings. | mini-chromosome maintenance (mcm) proteins form a conserved family found in all eukaryotes and are essential for dna replication. they exist as heteromultimeric complexes containing as many as six different proteins. these complexes are believed to be the replicative helicases, functioning as hexameric rings at replication forks. in most archaea a single mcm protein exists. the protein from methanobacterium thermoautotrophicum (mtmcm) has been reported to assemble into a large complex consistent ... | 2002 | 12151340 |
| human small intestinal mucosa harbours a small population of cytolytically active cd8+ alphabeta t lymphocytes. | intraepithelial lymphocytes (iel) in normal human small intestine exhibit cytotoxicity. this study was undertaken to characterize the effector cells and their mode of action. freshly isolated jejunal iel and lamina propria lymphocytes (lpl), as well as iel and lpl depleted of cd4+, cd8+ and t-cell receptor (tcr)-gammadelta+ cells were used as effector cells in anti-cd3-mediated redirected cytotoxicity against a murine fcgammar-expressing cell line. effector cell frequencies were estimated by eff ... | 2002 | 12153510 |
| 16s rrna mutation-mediated tetracycline resistance in helicobacter pylori. | most helicobacter pylori strains are susceptible to tetracycline, an antibiotic commonly used for the eradication of h. pylori. however, an increase in incidence of tetracycline resistance in h. pylori has recently been reported. here the mechanism of tetracycline resistance of the first dutch tetracycline-resistant (tet(r)) h. pylori isolate (strain 181) is investigated. twelve genes were selected from the genome sequences of h. pylori strains 26695 and j99 as potential candidate genes, based o ... | 2002 | 12183259 |
| the organization of cytoplasmic ribosomal protein genes in the arabidopsis genome. | eukaryotic ribosomes are made of two components, four ribosomal rnas, and approximately 80 ribosomal proteins (r-proteins). the exact number of r-proteins and r-protein genes in higher plants is not known. the strong conservation in eukaryotic r-protein primary sequence allowed us to use the well-characterized rat (rattus norvegicus) r-protein set to identify orthologues on the five haploid chromosomes of arabidopsis. by use of the numerous expressed sequence tag (est) accessions and the complet ... | 2001 | 11598216 |
| polypeptide release at sense and noncognate stop codons by localized charge-exchange alterations in translational release factors. | the mechanism of stop codon recognition during translation has long been a puzzle. only recently has it been established that a tripeptide in the bacterial release factors (rfs) 1 and 2 serves as the "anticodon" in deciphering stop codons in mrna. however, the molecular basis of the accuracy of stop codon recognition is unknown. although specific tripeptides in the rfs are primarily responsible for selective reading of cognate stop codons, charge-flip variant rf proteins, altered at conserved gl ... | 2002 | 11854484 |
| analysis of the aaa sensor-2 motif in the c-terminal atpase domain of hsp104 with a site-specific fluorescent probe of nucleotide binding. | hsp104 from saccharomyces cerevisiae is a hexameric protein with two aaa atpase domains (n- and c-terminal nucleotide-binding domains nbd1 and nbd2, respectively) per monomer. our previous analysis of the hsp104 atp hydrolysis cycle revealed that nbd1 and nbd2 have very different catalytic properties, but each shows positive cooperativity in hydrolysis. there is also communication between the two domains, in that atp hydrolysis at nbd1 depends on the nucleotide that is bound to nbd2. here, we ex ... | 2002 | 11867765 |
| novel domains and orthologues of eukaryotic transcription elongation factors. | the passage of rna polymerase ii across eukaryotic genes is impeded by the nucleosome, an octamer of histones h2a, h2b, h3 and h4 dimers. more than a dozen factors in the yeast saccharomyces cerevisiae are known to facilitate transcription elongation through chromatin. in order to better understand the evolution and function of these factors, their sequences have been compared with known protein, est and dna sequences. elongator subcomplex components elp4p and elp6p are shown to be homologues of ... | 2002 | 12202748 |
| influence of a sulfhydryl cross-link across the allosteric-site interface of e. coli phosphofructokinase. | to assess the role of quaternary stability on the properties of escherichia coli phosphofructokinase (pfk), a disulfide bond has been introduced across the subunit interface containing the allosteric binding sites in e. coli phosphofructokinase by changing n288 to cysteine. n288 is located in close proximity to the equivalent residue on an adjacent subunit. although sds-page of oxidized n288c indicates monomeric protein, blocking the six native cysteine residues with n-ethyl maleimide (nem) reve ... | 2001 | 11604525 |
| identification of a streptolysin s-associated gene cluster and its role in the pathogenesis of streptococcus iniae disease. | streptococcus iniae causes meningoencephalitis and death in cultured fish species and soft-tissue infection in humans. we recently reported that s. iniae is responsible for local tissue necrosis and bacteremia in a murine subcutaneous infection model. the ability to cause bacteremia in this model is associated with a genetic profile unique to strains responsible for disease in fish and humans (j. d. fuller, d. j. bast, v. nizet, d. e. low, and j. c. s. de azavedo, infect. immun. 69:1994-2000, 20 ... | 2002 | 12228303 |
| coordinated methyl and rna binding is required for heterochromatin localization of mammalian hp1alpha. | in mammalian cells, as in schizosaccharomyces pombe and drosophila, hp1 proteins bind histone h3 tails methylated on lysine 9 (k9). however, whereas k9-methylated h3 histones are distributed throughout the nucleus, hp1 proteins are enriched in pericentromeric heterochromatin. this observation suggests that the methyl-binding property of hp1 may not be sufficient for its heterochromatin targeting. we show that the association of hp1alpha with pericentromeric heterochromatin depends not only on it ... | 2002 | 12231507 |
| clue to damage recognition by uvrb: residues in the beta-hairpin structure prevent binding to non-damaged dna. | uvrb, the ultimate damage-recognizing component of bacterial nucleotide excision repair, contains a flexible beta-hairpin rich in hydrophobic residues. we describe the properties of uvrb mutants in which these residues have been mutated. the results show that y101 and f108 in the tip of the hairpin are important for the strand-separating activity of uvrb, supporting the model that the beta-hairpin inserts between the two dna strands during the search for dna damage. residues y95 and y96 at the b ... | 2001 | 11689453 |
| crystal structure of thermostable dna photolyase: pyrimidine-dimer recognition mechanism. | dna photolyase is a pyrimidine-dimer repair enzyme that uses visible light. photolyase generally contains two chromophore cofactors. one is a catalytic cofactor directly contributing to the repair of a pyrimidine-dimer. the other is a light-harvesting cofactor, which absorbs visible light and transfers energy to the catalytic cofactor. photolyases are classified according to their second cofactor into either a folate- or deazaflavin-type. the native structures of both types of photolyases have a ... | 2001 | 11707580 |
| saturation mutagenesis of 5s rrna in saccharomyces cerevisiae. | rrnas are the central players in the reactions catalyzed by ribosomes, and the individual rrnas are actively involved in different ribosome functions. our previous demonstration that yeast 5s rrna mutants (called mof9) can impact translational reading frame maintenance showed an unexpected function for this ubiquitous biomolecule. at the time, however, the highly repetitive nature of the genes encoding rrnas precluded more detailed genetic and molecular analyses. a new genetic system allows all ... | 2001 | 11713264 |
| overexpression, purification and characterization of recj protein from thermus thermophilus hb8 and its core domain. | a recj homolog was cloned from the extremely thermophilic bacterium thermus themophilus hb8. it encodes a 527 amino acid protein that has 33% identity to escherichia coli recj protein and includes the characteristic motifs conserved among recj homologs. although t.thermophilus recj protein (ttrecj) was expressed as an inclusion body, it was purified in soluble form through denaturation with urea and subsequent refolding steps. limited proteolysis showed that ttrecj has a protease-resistant core ... | 2001 | 11713311 |
| a novel type of uracil-dna glycosylase mediating repair of hydrolytic dna damage in the extremely thermophilic eubacterium thermus thermophilus. | spontaneous hydrolytic deamination of dna cytosine and 5-methyl-cytosine residues is an abundant source of c/g (5-mec/g) to t/a transition mutations. as a result of this pressure, at least six different families of enzymes have evolved that initiate repair at u/g (t/g) mispairs, the relevant pre-mutagenic intermediates. the necessarily higher rate of the process at elevated temperatures must pose a correspondingly accentuated problem to contemporary thermophilic organisms and may have been a ser ... | 2002 | 12000829 |
| proteomic characterization of the small subunit of chlamydomonas reinhardtii chloroplast ribosome: identification of a novel s1 domain-containing protein and unusually large orthologs of bacterial s2, s3, and s5. | to understand how chloroplast mrnas are translated into functional proteins, a detailed understanding of all of the components of chloroplast translation is needed. to this end, we performed a proteomic analysis of the plastid ribosomal proteins in the small subunit of the chloroplast ribosome from the green alga chlamydomonas reinhardtii. twenty proteins were identified, including orthologs of escherichia coli s1, s2, s3, s4, s5, s6, s7, s9, s10, s12, s13, s14, s15, s16, s17, s18, s19, s20, and ... | 2002 | 12417713 |
| mefloquine and new related compounds target the f(0) complex of the f(0)f(1) h(+)-atpase of streptococcus pneumoniae. | the activities of mefloquine (mfl) and related compounds against previously characterized streptococcus pneumoniae strains carrying defined amino acid substitutions in the c subunit of the f(0)f(1) h(+)-atpase were studied. in addition, a series of mfl-resistant (mfl(r)) strains were isolated and characterized. a good correlation was observed between inhibition of growth and inhibition of the membrane-associated f(0)f(1) h(+)-atpase activity. mfl was about 10-fold more active than optochin and a ... | 2002 | 12019076 |
| emergence of tetracycline resistance in helicobacter pylori: multiple mutational changes in 16s ribosomal dna and other genetic loci. | tetracycline is useful in combination therapies against the gastric pathogen helicobacter pylori. we found 6 tetracycline-resistant (tet(r)) strains among 159 clinical isolates (from el salvador, lithuania, and india) and obtained the following four results: (i) 5 of 6 tet(r) isolates contained one or two nucleotide substitutions in one part of the primary tetracycline binding site in 16s rrna (aga(965-967) [escherichia coli coordinates] changed to gga, agc, gua, or ggc [lowercase letters are us ... | 2002 | 12435699 |
| unique presence of a manganese catalase in a hyperthermophilic archaeon, pyrobaculum calidifontis va1. | we had previously isolated a facultatively anaerobic hyperthermophilic archaeon, pyrobaculum calidifontis strain va1. here, we found that strain va1, when grown under aerobic conditions, harbors high catalase activity. the catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 u/mg at 70 degrees c. the enzyme exhibited a k(m) value of 170 mm toward h(2)o(2) and a k(cat) value of 2.9 x 10(4) s(-1).subunit(-1) at 25 degrees c. gel filtration chromatography in ... | 2002 | 12029047 |
| thermoadaptation of alpha-galactosidase agab1 in thermus thermophilus. | the evolutionary potential of a thermostable alpha-galactosidase, with regard to improved catalytic activity at high temperatures, was investigated by employing an in vivo selection system based on thermophilic bacteria. for this purpose, hybrid alpha-galactosidase genes of agaa and agab from bacillus stearothermophilus kve39, designated agaa1 and agab1, were cloned into an autonomously replicating thermus vector and introduced into thermus thermophilus of1053gd (deltaagat) by transformation. th ... | 2002 | 12029056 |
| elucidation of structure-function relationships in the protein subunit of bacterial rnase p using a genetic complementation approach. | rnase p is a ribonucleoprotein involved in trna biosynthesis in all living organisms. bacterial rnase p is comprised of a catalytic rna subunit and a lone protein cofactor which plays a supporting, albeit essential, role in the trna processing reaction in vivo. in this study, we have searched various databases to identify homologs of the protein subunit of rnase p from diverse bacteria and used an alignment of their primary sequences to determine the most highly conserved residues, and thereby e ... | 2002 | 12466529 |
| structural origins of amino acid selection without editing by cysteinyl-trna synthetase. | cysteinyl-trna synthetase (cysrs) is highly specific for synthesis of cysteinyl adenylate, yet does not possess the amino acid editing activity characteristic of many other trna synthetases. to elucidate how cysrs is able to distinguish cysteine from non-cognate amino acids, crystal structures of the escherichia coli enzyme were determined in apo and cysteine-bound states. the structures reveal that the substrate cysteine thiolate forms a single direct interaction with a zinc ion bound at the ba ... | 2002 | 12032090 |
| in vivo selection of spectinomycin-binding rnas. | the folding of even short rna molecules in a random library can produce a huge number of possible macromolecular structures. using this principle, we have designed selections to seek non-coding rna transcripts capable of interfering with specific macromolecules such as transcription factors in living bacterial cells. here we show that such selections can uncover an unexpected class of rnas. in the present case, we report short rna transcripts whose expression confers bacterial resistance to the ... | 2002 | 12490711 |
| characterization of the interaction between the nucleotide exchange factor ef-ts from nematode mitochondria and elongation factor tu. | caenorhabditis elegans mitochondria have two elongation factor (ef)-tu species, denoted ef-tu1 and ef-tu2. recombinant nematode ef-ts purified from escherichia coli bound both of these molecules and also stimulated the translational activity of ef-tu, indicating that the nematode ef-ts homolog is a functional ef-ts protein of mitochondria. complexes formed by the interaction of nematode ef-ts with ef-tu1 and ef-tu2 could be detected by native gel electrophoresis and purified by gel filtration. a ... | 2002 | 12490713 |
| structure of the topoisomerase vi-b subunit: implications for type ii topoisomerase mechanism and evolution. | type iia and type iib topoisomerases each possess the ability to pass one dna duplex through another in an atp-dependent manner. the role of atp in the strand passage reaction is poorly understood, particularly for the type iib (topoisomerase vi) family. we have solved the structure of the atp-binding subunit of topoisomerase vi (topovi-b) in two states: an unliganded monomer and a nucleotide-bound dimer. we find that topovi-b is highly structurally homologous to the entire 40-43 kda atpase regi ... | 2003 | 12505993 |
| role of ctc from listeria monocytogenes in osmotolerance. | listeria monocytogenes is a food-borne pathogen with the ability to grow under conditions of high osmolarity. in a previous study, we reported the identification of 12 proteins showing high induction after salt stress. one of these proteins is highly similar to the general stress protein ctc of bacillus subtilis. in this study, induction of ctc after salt stress was confirmed at the transcriptional level by using rna slot blot experiments. to explore the role of the ctc gene product in resistanc ... | 2003 | 12513990 |
| detailed analysis of rna-protein interactions within the bacterial ribosomal protein l5/5s rrna complex. | the crystal structure of ribosomal protein l5 from thermus thermophilus complexed with a 34-nt fragment comprising helix iii and loop c of escherichia coli 5s rrna has been determined at 2.5 a resolution. the protein specifically interacts with the bulged nucleotides at the top of loop c of 5s rrna. the rrna and protein contact surfaces are strongly stabilized by intramolecular interactions. charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two ... | 2002 | 12515387 |
| uptake and antifungal activity of oligonucleotides in candida albicans. | candida albicans is a significant cause of disease in immunocompromised humans. because the number of people infected by fungal pathogens is increasing, strategies are being developed to target rnas in fungi. this work shows that oligonucleotides can serve as therapeutics against c. albicans. in particular, oligonucleotides are taken up from cell culture medium in an energy-dependent process. after uptake, oligonucleotides, including rna, remain mostly intact after 12 h in culture. for culture c ... | 2003 | 12552085 |
| atp binding by glutamyl-trna synthetase is switched to the productive mode by trna binding. | aminoacyl-trna synthetases catalyze the formation of an aminoacyl-amp from an amino acid and atp, prior to the aminoacyl transfer to trna. a subset of aminoacyl-trna synthetases, including glutamyl-trna synthetase (glurs), have a regulation mechanism to avoid aminoacyl-amp formation in the absence of trna. in this study, we determined the crystal structure of the 'non-productive' complex of thermus thermophilus glurs, atp and l-glutamate, together with those of the glurs.atp, glurs.trna.atp and ... | 2003 | 12554668 |
| importance of compartment formation for a self-encoding system. | a self-encoding system designed to have strict "compartition" of the molecules, i.e., to contain only a single molecule of dna in each compartment, was established, and its evolutionary fate was analyzed. the system comprised the thermus thermophilus dna polymerase gene as the informational molecule and its protein product replicating the gene as the functional molecule. imposing strict compartition allows the self-encoding system to last up to at least the tenth generation, whereas the system c ... | 2002 | 12032314 |
| mechanism of molecular interactions for trna(val) recognition by valyl-trna synthetase. | the molecular interactions between valyl-trna synthetase (valrs) and trna(val), with the c34-a35-c36 anticodon, from thermus thermophilus were studied by crystallographic analysis and structure-based mutagenesis. in the valrs-bound structure of trna(val), the successive a35-c36 residues (the major identity elements) of trna(val) are base-stacked upon each other, and fit into a pocket on the alpha-helix bundle domain of valrs. hydrogen bonds are formed between valrs and a35-c36 of trna(val) in a ... | 2003 | 12554880 |
| transfer rna determinants for translational editing by escherichia coli valyl-trna synthetase. | valyl-trna synthetase (valrs) has difficulty differentiating valine from structurally similar non-cognate amino acids, most prominently threonine. to minimize errors in aminoacylation and translation the enzyme catalyzes a proofreading (editing) reaction that is dependent on the presence of cognate trna(val). editing occurs at a site functionally distinct from the aminoacylation site of valrs and previous results have shown that the 3'-terminus of trna(val) is recognized differently at the two s ... | 2002 | 12034843 |
| a novel uracil-dna glycosylase with broad substrate specificity and an unusual active site. | uracil-dna glycosylases (udgs) catalyse the removal of uracil by flipping it out of the double helix into their binding pockets, where the glycosidic bond is hydrolysed by a water molecule activated by a polar amino acid. interestingly, the four known udg families differ in their active site make-up. the activating residues in ung and smug enzymes are aspartates, thermostable udgs resemble ung-type enzymes, but carry glutamate rather than aspartate residues in their active sites, and the less ac ... | 2002 | 12065430 |
| evidence for rotation of v1-atpase. | v(o)v(1)-atpase is responsible for acidification of eukaryotic intracellular compartments and atp synthesis of archaea and some eubacteria. from the similarity to f(o)f(1)-atp synthase, v(o)v(1)-atpase has been assumed to be a rotary motor, but to date there are no experimental data to support this. here we visualized the rotation of single molecules of v(1)-atpase, a catalytic subcomplex of v(o)v(1)-atpase. v(1)-atpase from thermus thermophilus was immobilized onto a glass surface, and a bead w ... | 2003 | 12598655 |
| the identification of spermine binding sites in 16s rrna allows interpretation of the spermine effect on ribosomal 30s subunit functions. | a photoreactive analogue of spermine, n1-azidobenzamidino (aba)-spermine, was covalently attached after irradiation to escherichia coli 30s ribosomal subunits or naked 16s rrna. by means of rnase h digestion and primer extension, the cross-linking sites of aba-spermine in naked 16s rrna were characterised and compared with those identified in 30s subunits. the 5' domain, the internal and terminal loops of helix h24, as well as the upper part of helix h44 in naked 16s rrna, were found to be prefe ... | 2002 | 12087167 |
| genetic diversity: frameshift mechanisms alter coding of a gene (epstein-barr virus lf3 gene) that contains multiple 102-base-pair direct sequence repeats. | frameshift mutations provide recognized mechanisms for changing the coding potential of an organism. here, multiple frameshifts are identified in repetitive sequences within an epstein-barr virus unspliced early gene, lf3, which is associated with the viral replicative cycle and also transcriptionally expressed in many virally associated tumors. on the dna strand encoding lf3, there are three open reading frames, only one of which contains an initiation codon. most (>95%) of the gene consists of ... | 2003 | 12612089 |
| the feci extracytoplasmic-function sigma factor of escherichia coli interacts with the beta' subunit of rna polymerase. | transcription of the ferric citrate transport system of escherichia coli k-12 is mediated by the extracytoplasmic-function (ecf) sigma factor feci, which is activated by ferric citrate in the growth medium. by using a bacterial two-hybrid system, it was shown in vivo that feci binds to the beta' subunit of rna polymerase. the inactive mutant protein feci(k155e) displayed reduced binding to beta', and small deletions along the entire feci protein led to total impairment of beta' binding. in vitro ... | 2003 | 12618442 |
| molecular characterization of the s-layer gene, sbpa, of bacillus sphaericus ccm 2177 and production of a functional s-layer fusion protein with the ability to recrystallize in a defined orientation while presenting the fused allergen. | the nucleotide sequence encoding the crystalline bacterial cell surface (s-layer) protein sbpa of bacillus sphaericus ccm 2177 was determined by a pcr-based technique using four overlapping fragments. the entire sbpa sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 da and a calculated isoelectric point of 4.69. the n-terminal part of sbpa, which is involved in anchoring the s-layer subunits via a distinct t ... | 2002 | 12089001 |
| stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection. | prions are composed of an isoform of a normal sialoglycoprotein called prp(c), whose physiological role has been under investigation, with focus on the screening for ligands. our group described a membrane 66 kda prp(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 ... | 2002 | 12093732 |
| characterization of a novel thermostable o-acetylserine sulfhydrylase from aeropyrum pernix k1. | an o-acetylserine sulfhydrylase (oass) from the hyperthermophilic archaeon aeropyrum pernix k1, which shares the pyridoxal 5'-phosphate binding motif with both oass and cystathionine beta-synthase (cbs), was cloned and expressed by using escherichia coli rosetta(de3). the purified protein was a dimer and contained pyridoxal 5'-phosphate. it was shown to be an enzyme with cbs activity as well as oass activity in vitro. the enzyme retained 90% of its activity after a 6-h incubation at 100 degrees ... | 2003 | 12644499 |
| antagonistic signals within the cox2 mrna coding sequence control its translation in saccharomyces cerevisiae mitochondria. | translation of the mitochondrially coded cox2 mrna within the organelle in yeast produces the precursor of cox2p (pre-cox2p), which is processed and assembled into cytochrome c oxidase. the mrna sequence of the first 14 cox2 codons, specifying the pre-cox2p leader peptide, was previously shown to contain a positively acting element required for translation of a mitochondrial reporter gene, arg8(m), fused to the 91st codon of cox2. here we show that three relatively short sequences within the cox ... | 2003 | 12649494 |
| the trna specificity of thermus thermophilus ef-tu. | by introducing a gac anticodon, 21 different escherichia coli trnas were misacylated with either phenylalanine or valine and assayed for their affinity to thermus thermophilus elongation factor tu (ef-tu)*gtp by using a ribonuclease protection assay. the presence of a common esterified amino acid permits the thermodynamic contribution of each trna body to the overall affinity to be evaluated. the e. coli elongator trnas exhibit a wide range of binding affinities that varied from -11.7 kcal/mol f ... | 2002 | 11891293 |