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three crystal forms of the bifunctional enzyme proline utilization a (puta) from bradyrhizobium japonicum.proline utilization a proteins (putas) are large (1000-1300 residues) membrane-associated bifunctional flavoenzymes that catalyze the two-step oxidation of proline to glutamate by the sequential action of proline dehydrogenase and delta(1)-pyrroline-5-carboxylate dehydrogenase domains. here, the first successful crystallization efforts for a puta protein are described. three crystal forms of puta from bradyrhizobium japonicum are reported: apparent tetragonal, hexagonal and centered monoclinic. ...200818931443
rna folding and ribosome assembly.ribosome synthesis is a tightly regulated process that is crucial for cell survival. chemical footprinting, mass spectrometry, and cryo-electron microscopy are revealing how these complex cellular machines are assembled. rapid folding of the rrna provides a platform for protein-induced assembly of the bacterial 30s ribosome. multiple assembly pathways increase the flexibility of the assembly process, while accessory factors and modification enzymes chaperone the late stages of assembly and contr ...200818935976
path of nascent polypeptide in exit tunnel revealed by molecular dynamics simulation of ribosome.molecular dynamics simulations were carried out on thermus thermophilus 70s ribosome with and without a nascent polypeptide inside the exit tunnel. modeling of the polypeptide in the tunnel revealed two possible paths: one over arg92 of l22 and one under (from the viewpoint of 50s on top of 30s). a strong interaction between l4 and arg92 was observed without the polypeptide and when it passed over arg92. however, when the polypeptide passed under, arg92 repositioned to interact with ade2059 of 2 ...200818936244
ssb as an organizer/mobilizer of genome maintenance complexes.when duplex dna is altered in almost any way (replicated, recombined, or repaired), single strands of dna are usually intermediates, and single-stranded dna binding (ssb) proteins are present. these proteins have often been described as inert, protective dna coatings. continuing research is demonstrating a far more complex role of ssb that includes the organization and/or mobilization of all aspects of dna metabolism. escherichia coli ssb is now known to interact with at least 14 other proteins ...200818937104
a full-length group 1 bacterial sigma factor adopts a compact structure incompatible with dna binding.the sigma factors are the key regulators of bacterial transcription initiation. through direct read-out of promoter dna sequence, they recruit the core rna polymerase to sites of initiation, thereby dictating the rna polymerase promoter-specificity. the group 1 sigma factors, which direct the vast majority of transcription initiation during log phase growth and are essential for viability, are autoregulated by an n-terminal sequence known as sigma1.1. we report the solution structure of thermoto ...200818940669
structural basis for specific, high-affinity tetracycline binding by an in vitro evolved aptamer and artificial riboswitch.the tetracycline aptamer is an in vitro selected rna that binds to the antibiotic with the highest known affinity of an artificial rna for a small molecule (kd approximately 0.8 nm). it is one of few aptamers known to be capable of modulating gene expression in vivo. the 2.2 a resolution cocrystal structure of the aptamer reveals a pseudoknot-like fold formed by tertiary interactions between an 11 nucleotide loop and the minor groove of an irregular helix. tetracycline binds within this interfac ...200818940672
glycine cleavage system: reaction mechanism, physiological significance, and hyperglycinemia.the glycine cleavage system catalyzes the following reversible reaction: glycine + h(4)folate + nad(+) <==> 5,10-methylene-h(4)folate + co(2) + nh(3) + nadh + h(+)the glycine cleavage system is widely distributed in animals, plants and bacteria and consists of three intrinsic and one common components: those are i) p-protein, a pyridoxal phosphate-containing protein, ii) t-protein, a protein required for the tetrahydrofolate-dependent reaction, iii) h-protein, a protein that carries the aminomet ...200818941301
ruva and ruvb mutants specifically impaired for replication fork reversal.replication fork reversal (rfr) is a reaction that takes place in escherichia coli at replication forks arrested by the inactivation of a replication protein. fork reversal involves the annealing of the leading and lagging strand ends; it results in the formation of a holliday junction adjacent to dna double-strand end, both of which are processed by recombination enzymes. in several replication mutants, replication fork reversal is catalysed by the ruvab complex, originally characterized for it ...200818942176
higher-order association states of cellular erbb3 probed with photo-cross-linkable aptamers.nucleic acid aptamers are rapidly gaining prominence as diagnostic tools, targeting reagents, and potential therapeutics. to extend the use of aptamers into the biochemical analysis of protein interactions on the surface of live cells, we converted an enzymatically generated rna aptamer into a photo-cross-linkable affinity tag through the replacement of all uracils with 4-thiouracil. specifically, we converted a previously selected, inhibitory aptamer that binds the soluble extracellular domains ...200818942860
side-chain recognition and gating in the ribosome exit tunnel.the ribosome is a large complex catalyst responsible for the synthesis of new proteins, an essential function for life. new proteins emerge from the ribosome through an exit tunnel as nascent polypeptide chains. recent findings indicate that tunnel interactions with the nascent polypeptide chain might be relevant for the regulation of translation. however, the specific ribosomal structural features that mediate this process are unknown. performing molecular dynamics simulations, we are studying ...200818946046
transcription inactivation through local refolding of the rna polymerase structure.structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. myxopyronin inhibits bacterial rna polymerase (rnap) by an unknown mechanism. here we report the structure of dmyx--a desmethyl derivative of myxopyronin b--complexed with a thermus thermophilus rnap holoenzyme. the antibiotic binds to a pocket deep inside the rnap clamp h ...200918946472
comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting saccharomyces cerevisiae strains.ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. this process would greatly benefit from recombinant saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. different pathways can be introduced in s. cerevisiae to provide arabinose and xylose utilisation. in this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation p ...200818947407
genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world.the first bacterial genome was sequenced in 1995, and the first archaeal genome in 1996. soon after these breakthroughs, an exponential rate of genome sequencing was established, with a doubling time of approximately 20 months for bacteria and approximately 34 months for archaea. comparative analysis of the hundreds of sequenced bacterial and dozens of archaeal genomes leads to several generalizations on the principles of genome organization and evolution. a crucial finding that enables function ...200818948295
structure of the yeast vacuolar atpase.the subunit architecture of the yeast vacuolar atpase (v-atpase) was analyzed by single particle transmission electron microscopy and electrospray ionization (esi) tandem mass spectrometry. a three-dimensional model of the intact v-atpase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. images of yeast v-atpase decorated with monoclonal antibodies against subunits a, e, and g position subunit a within the pseudo-hexagonal arrangement in the v1, the ...200818955482
tie me up, tie me down: inhibiting rna polymerase.mechanistic understanding of antibiotic action can yield crucial insights that aid in the design of new antibiotics. in this issue, mukhopadhyay et al. (2008) uncover the mechanism by which the antibiotic myxopyronin inhibits bacterial rna polymerase, suggesting a new target region in rna polymerase for inhibitor design.200818957193
the p. furiosus mre11/rad50 complex promotes 5' strand resection at a dna double-strand break.the mre11/rad50 complex has been implicated in the early steps of dna double-strand break (dsb) repair through homologous recombination in several organisms. however, the enzymatic properties of this complex are incompatible with the generation of 3' single-stranded dna for recombinase loading and strand exchange. in thermophilic archaea, the mre11 and rad50 genes cluster in an operon with genes encoding a helicase, hera, and a 5' to 3' exonuclease, nura, suggesting a common function. here we sh ...200818957200
the rna polymerase "switch region" is a target for inhibitors.the alpha-pyrone antibiotic myxopyronin (myx) inhibits bacterial rna polymerase (rnap). here, through a combination of genetic, biochemical, and structural approaches, we show that myx interacts with the rnap "switch region"--the hinge that mediates opening and closing of the rnap active center cleft--to prevent interaction of rnap with promoter dna. we define the contacts between myx and rnap and the effects of myx on rnap conformation and propose that myx functions by interfering with opening ...200818957204
rna in motion.although rna duplex regions are highly structured and inflexible, other elements of an rna molecule are capable of dynamic motions. these flexible regions are the sites of interactions with small molecules, proteins, and other rnas, yet there are few descriptions of these regions that include the timescale and amplitude of their motions. no one technique is sufficient to accurately describe these motions, but the combination of in vitro methods, particularly nmr relaxation methods, and more robu ...200818957331
automated motif extraction and classification in rna tertiary structures.we used a novel graph-based approach to extract rna tertiary motifs. we cataloged them all and clustered them using an innovative graph similarity measure. we applied our method to three widely studied structures: haloarcula marismortui 50s (h.m 50s), escherichia coli 50s (e. coli 50s), and thermus thermophilus 16s (t.th 16s) rnas. we identified 10 known motifs without any prior knowledge of their shapes or positions. we additionally identified four putative new motifs.200818957493
sequence and structural evolution of the ksga/dim1 methyltransferase family.one of the 60 or so genes conserved in all domains of life is the ksga/dim1 orthologous group. enzymes from this family perform the same post-transcriptional nucleotide modification in ribosome biogenesis, irrespective of organism. despite this common function, divergence has enabled some family members to adopt new and sometimes radically different functions. for example, in s. cerevisiae dim1 performs two distinct functions in ribosome biogenesis, while human mttfb is not only an rrna methyltr ...200818959795
cytochrome c oxidase: exciting progress and remaining mysteries.cytochrome c oxidase generates a proton motive force by two separate mechanisms. the first mechanism is similar to that postulated by peter mitchell, and is based on electrons and protons used to generate water coming from opposite sides of the membrane. the second mechanism was not initially anticipated, but is now firmly established as a proton pump. a brief review of the current state of our understanding of the proton pump of cytochrome oxidase is presented. we have come a long way since the ...200818975062
translation initiation factor if1 of bacillus stearothermophilus and thermus thermophilus substitute for escherichia coli if1 in vivo and in vitro without a direct if1-if2 interaction.bacterial translation initiation factor if1 is homologous to archaeal aif1a and eukaryal eif1a, which form a complex with their homologous if2-like factors (aif5b and eif5b respectively) during initiation of protein synthesis. a similar if1-if2 interaction is assumed to occur in all bacteria and supported by cross-linking data and stabilization of the 30s-if2 interaction by if1. here we compare escherichia coli if1 with thermophilic factors from bacillus stearothermophilus and thermus thermophil ...200818976282
bacterial heme-transport proteins and their heme-coordination modes.efficient iron acquisition is critical for an invading microbe's survival and virulence. most of the iron in mammals is incorporated into heme, which can be plundered by certain bacterial pathogens as a nutritional iron source. utilization of exogenous heme by bacteria involves the binding of heme or hemoproteins to the cell surface receptors, followed by the transport of heme into cells. once taken into the cytosol, heme is presented to heme oxygenases where the tetrapyrrole ring is cleaved in ...200818977196
bacterial heme-transport proteins and their heme-coordination modes.efficient iron acquisition is critical for an invading microbe's survival and virulence. most of the iron in mammals is incorporated into heme, which can be plundered by certain bacterial pathogens as a nutritional iron source. utilization of exogenous heme by bacteria involves the binding of heme or hemoproteins to the cell surface receptors, followed by the transport of heme into cells. once taken into the cytosol, heme is presented to heme oxygenases where the tetrapyrrole ring is cleaved in ...200818977196
unfolding thermodynamics of the delta-domain in the prohead i subunit of phage hk97: determination by factor analysis of raman spectra.an early step in the morphogenesis of the double-stranded dna (dsdna) bacteriophage hk97 is the assembly of a precursor shell (prohead i) from 420 copies of a 384-residue subunit (gp5). although formation of prohead i requires direct participation of gp5 residues 2-103 (delta-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and dna packaging. the prohead i delta-domain is thought to resemble a phage scaffolding protein, by virtue of its highly alpha-helic ...200918983851
unfolding thermodynamics of the delta-domain in the prohead i subunit of phage hk97: determination by factor analysis of raman spectra.an early step in the morphogenesis of the double-stranded dna (dsdna) bacteriophage hk97 is the assembly of a precursor shell (prohead i) from 420 copies of a 384-residue subunit (gp5). although formation of prohead i requires direct participation of gp5 residues 2-103 (delta-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and dna packaging. the prohead i delta-domain is thought to resemble a phage scaffolding protein, by virtue of its highly alpha-helic ...200918983851
recognition of a common rdna target site in archaea and eukarya by analogous laglidadg and his-cys box homing endonucleases.the presence of a homing endonuclease gene (heg) within a microbial intron or intein empowers the entire element with the ability to invade genomic targets. the persistence of a homing endonuclease lineage depends in part on conservation of its dna target site. one such rdna sequence has been invaded both in archaea and in eukarya, by laglidadg and his-cys box homing endonucleases, respectively. the bases encoded by this target include a universally conserved ribosomal structure, termed helix 69 ...200818984620
insights into translational termination from the structure of rf2 bound to the ribosome.the termination of protein synthesis occurs through the specific recognition of a stop codon in the a site of the ribosome by a release factor (rf), which then catalyzes the hydrolysis of the nascent protein chain from the p-site transfer rna. here we present, at a resolution of 3.5 angstroms, the crystal structure of rf2 in complex with its cognate uga stop codon in the 70s ribosome. the structure provides insight into how rf2 specifically recognizes the stop codon; it also suggests a model for ...200818988853
organization of an activator-bound rna polymerase holoenzyme.transcription initiation involves the conversion from closed promoter complexes, comprising rna polymerase (rnap) and double-stranded promoter dna, to open complexes, in which the enzyme is able to access the dna template in a single-stranded form. the complex between bacterial rnap and its major variant sigma factor sigma(54) remains as a closed complex until atp hydrolysis-dependent remodeling by activator proteins occurs. this remodeling facilitates dna melting and allows the transition to th ...200818995832
rv0802c from mycobacterium tuberculosis: the first structure of a succinyltransferase with the gnat fold.gene rv0802c from mycobacterium tuberculosis encodes a 218-amino-acid protein and is annotated as a hypothetical protein with homology to gcn5-related n-acetyltransferases. the structure of rv0802c was determined in an unliganded form to 2.0 a resolution utilizing single-wavelength anomalous dispersion from a samarium soak that resulted in a single bound sm(3+):citrate(2) complex. the structure confirms that rv0802c exhibits the gcn5-related n-acetyltransferase fold and revealed a tetramer compo ...200818997321
core structure of the yeast spt4-spt5 complex: a conserved module for regulation of transcription elongation.the spt4-spt5 complex is an essential rna polymerase ii elongation factor found in all eukaryotes and important for gene regulation. we report here the crystal structure of saccharomyces cerevisiae spt4 bound to the ngn domain of spt5. this structure reveals that spt4-spt5 binding is governed by an acid-dipole interaction between spt5 and spt4. mutations that disrupt this interaction disrupt the complex. residues forming this pivotal interaction are conserved in the archaeal homologs of spt4 and ...200819000817
escherichia coli tmrna lacking pseudoknot 1 tags truncated proteins in vivo and in vitro.transfer-messenger rna (tmrna) and protein smpb facilitate trans-translation, a quality-control process that tags truncated proteins with short peptides recognized by a number of proteases and recycles ribosomes stalled at the 3' end of mrna templates lacking stop codons. the tmrna molecule is a hybrid of trna- and mrna-like domains that are usually connected by four pseudoknots (pk1-pk4). replacement of pk1 with a single-stranded rna yields pk1l, a mutant tmrna that tags truncated proteins very ...200919001120
the hsp70 chaperone system maintains high concentrations of active proteins and suppresses atp consumption during heat shock.hsp70 chaperones assist protein folding by cycling between the atp-bound t state with low affinity for substrates and the adp-bound r state with high affinity for substrates. the transition from the t to r state is catalyzed by the synergistic action of the substrate and dnaj cochaperones. the reverse transition from the r state to the t state is accelerated by the nucleotide exchange factor grpe. these two processes, t-to-r and r-to-t conversion, are affected differently by temperature change. ...200719003436
collective dynamics of the ribosomal tunnel revealed by elastic network modeling.the collective dynamics of the nascent polypeptide exit tunnel are investigated with the computationally efficient elastic network model using normal mode analysis. the calculated normal modes are considered individually and in linear combinations with different coefficients mimicking the phase angles between modes, in order to follow the mechanistic motions of tunnel wall residues. the low frequency fluctuations indicate three distinct regions along the tunnel-the entrance, the neck, and the ex ...200919004020
high tolerance for ionizable residues in the hydrophobic interior of proteins.internal ionizable groups are quite rare in water-soluble globular proteins. presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. here we show that proteins can have an inherently high tolerance for internal ionizable groups. the 25 internal positions in staphylococcal nuclease were substituted one at a time with lys, glu, or asp without abolishing enzymatic activity and without detectable changes in the conformation of the protei ...200819004768
probing the paracoccus denitrificans cytochrome c(1)-cytochrome c(552) interaction by mutagenesis and fast kinetics.electron transfer (et) between paracoccus denitrificans cytochrome (cyt) c(1) and cytochrome c(552) was studied using the soluble redox fragments cyt c(1cf) and cyt c(552f). a new ruthenium cyt c(552f) derivative labeled at c23 (ru(z)-23-c(552f)) was designed to measure rapid electron transfer with cyt c(1cf) in the physiological direction using flash photolysis. the bimolecular rate constant k(12) decreased rapidly with ionic strength above 40 mm, consistent with a diffusional process guided by ...200819006325
a short-oligonucleotide microarray that allows improved detection of gastrointestinal tract microbial communities.the human gastrointestinal (gi) tract contains a diverse collection of bacteria, most of which are unculturable by conventional microbiological methods. increasingly molecular profiling techniques are being employed to examine this complex microbial community. the purpose of this study was to develop a microarray technique based on 16s ribosomal gene sequences for rapidly monitoring the microbial population of the gi tract.200819014434
prevalence of pfmdr1, pfcrt, pfdhfr and pfdhps mutations associated with drug resistance, in luanda, angola.malaria is the infectious disease causing the highest morbidity and mortality in angola and due to widespread chloroquine (cq) resistance, the country has recently changed its first-line treatment recommendations for uncomplicated malaria, from cq to artemisinin combination therapies (act) in adults, and sulphadoxine/pyrimethamine (s/p) in pregnant women. loss of sp sensitivity is, however, progressing rapidly in africa and, in this study, were investigated a number of molecular markers associat ...200819014684
contributions of the two accessory subunits, rnaseh2b and rnaseh2c, to the activity and properties of the human rnase h2 complex.eukaryotic rnase h2 is a heterotrimeric enzyme. here, we show that the biochemical composition and stoichiometry of the human rnase h2 complex is consistent with the properties previously deduced from genetic studies. the catalytic subunit of eukaryotic rnase h2, rnaseh2a, is well conserved and similar to the monomeric prokaryotic rnase hii. in contrast, the rnaseh2b and rnaseh2c subunits from human and saccharomyces cerevisiae share very little homology, although they both form soluble b/c comp ...200919015152
contributions of the two accessory subunits, rnaseh2b and rnaseh2c, to the activity and properties of the human rnase h2 complex.eukaryotic rnase h2 is a heterotrimeric enzyme. here, we show that the biochemical composition and stoichiometry of the human rnase h2 complex is consistent with the properties previously deduced from genetic studies. the catalytic subunit of eukaryotic rnase h2, rnaseh2a, is well conserved and similar to the monomeric prokaryotic rnase hii. in contrast, the rnaseh2b and rnaseh2c subunits from human and saccharomyces cerevisiae share very little homology, although they both form soluble b/c comp ...200919015152
structural and biochemical studies of tigar (tp53-induced glycolysis and apoptosis regulator).activation of the p53 tumor suppressor by cellular stress leads to variable responses ranging from growth inhibition to apoptosis. tigar is a novel p53-inducible gene that inhibits glycolysis by reducing cellular levels of fructose-2,6-bisphosphate, an activator of glycolysis and inhibitor of gluconeogenesis. here we describe structural and biochemical studies of tigar from danio rerio. the overall structure forms a histidine phosphatase fold with a phosphate molecule coordinated to the catalyti ...200919015259
revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein l22.bacterial antibiotic resistance can occur by many mechanisms. an intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. for example, a 3-residue deletion (deltamkr) in ribosomal protein l22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. here, however, we demonstrate that deltamkr and wild-type ribosomes show comparab ...200819015512
probing protein structure by amino acid-specific covalent labeling and mass spectrometry.for many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. these covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. the reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label ...200919016300
transposition of an insertion sequence, istth7, in the genome of the extreme thermophile thermus thermophilus hb8.we have identified an active insertion sequence (is) in the genome of thermus thermophilus hb8. transposition was detected as insertional inactivation of a 16s rrna methyltransferase gene, rsmg, resulting in streptomycin resistance. the is element, istth7, is 1029 bp in length, encodes an imperfect 12 bp inverted repeat, and produces a 9 bp direct repeat of the target sequence. the sequence of a putative transposase encoded by istth7 indicates that it is a member of the is427 group within the is ...200819016874
recr-mediated modulation of recf dimer specificity for single- and double-stranded dna.recf pathway proteins play an important role in the restart of stalled replication and dna repair in prokaryotes. following dna damage, recf, recr, and reco initiate homologous recombination (hr) by loading of the reca recombinase on single-stranded (ss) dna, protected by ssdna-binding protein. the specific role of recf in this process is not well understood. previous studies have proposed that recf directs the recor complex to boundaries of damaged dna regions by recognizing single-stranded/dou ...200919017635
novel escherichia coli rf1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins kid and rele.novel mutations in prfa, the gene for the polypeptide release factor rf1 of escherichia coli, were isolated using a positive genetic screen based on the pard (kis, kid) toxin-antitoxin system. this original approach allowed the direct selection of mutants with altered translational termination efficiency at uag codons. the isolated prfa mutants displayed a approximately 10-fold decrease in uag termination efficiency with no significant changes in rf1 stability in vivo. all three mutations, g121s ...200919019162
genome-wide survey of prokaryotic serine proteases: analysis of distribution and domain architectures of five serine protease families in prokaryotes.serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. while studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown. current analysis is aimed at understanding the distribution and probable b ...200819019219
phylogenetic analysis of rubella virus strains from an outbreak in madrid, spain, from 2004 to 2005.an outbreak of rubella affected 460 individuals in 2004 and 2005 in the community of madrid, spain. most of the patients were nonvaccinated latin american immigrants or spanish males. this study presents the first data on rubella virus genotypes in spain. forty selected clinical samples (2 urine, 5 serum, 3 blood, 2 saliva, and 28 pharyngeal exudate samples) from 40 cases were collected. the 739-nucleotide sequence recommended by the world health organization obtained from viral rna in these sam ...200919020066
phylogenetic analysis of rubella virus strains from an outbreak in madrid, spain, from 2004 to 2005.an outbreak of rubella affected 460 individuals in 2004 and 2005 in the community of madrid, spain. most of the patients were nonvaccinated latin american immigrants or spanish males. this study presents the first data on rubella virus genotypes in spain. forty selected clinical samples (2 urine, 5 serum, 3 blood, 2 saliva, and 28 pharyngeal exudate samples) from 40 cases were collected. the 739-nucleotide sequence recommended by the world health organization obtained from viral rna in these sam ...200919020066
recognition of aminoacyl-trna: a common molecular mechanism revealed by cryo-em.the accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by ef-tu, which forms a ternary complex with aminoacyl(aa)-trna. to study the binding modes of different aa-trnas, we compared cryo-em maps of the kirromycin-stalled ribosome bound with ternary complexes containing phe-trna(phe), trp-trna(trp), or leu-trna(leui). the three maps suggest a common binding manner of cognate aa-trnas in their specific binding with both the ribosome and ef-tu. a ...200819020518
structure and function of hiv-1 reverse transcriptase: molecular mechanisms of polymerization and inhibition.the rapid replication of hiv-1 and the errors made during viral replication cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly challenging. in the absence of an effective vaccine, drugs are the only useful treatment. anti-hiv drugs work; so far drug therapy has saved more than three million years of life. unfortunately, hiv-1 develops resistance to all of the available drugs. although a number of useful anti-hiv drugs have been ...200919022262
structure and function of hiv-1 reverse transcriptase: molecular mechanisms of polymerization and inhibition.the rapid replication of hiv-1 and the errors made during viral replication cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly challenging. in the absence of an effective vaccine, drugs are the only useful treatment. anti-hiv drugs work; so far drug therapy has saved more than three million years of life. unfortunately, hiv-1 develops resistance to all of the available drugs. although a number of useful anti-hiv drugs have been ...200919022262
glucose- and glucokinase-controlled mal gene expression in escherichia coli.malt is the central transcriptional activator of all mal genes in escherichia coli. its activity is controlled by the inducer maltotriose. it can be inhibited by the interaction with certain proteins, and its expression can be controlled. we report here a novel aspect of mal gene regulation: the effect of cytoplasmic glucose and glucokinase (glk) on the activity and the expression of malt. amylomaltase (malq) is essential for the metabolism of maltose. it forms maltodextrins and glucose from mal ...200919028900
glucose- and glucokinase-controlled mal gene expression in escherichia coli.malt is the central transcriptional activator of all mal genes in escherichia coli. its activity is controlled by the inducer maltotriose. it can be inhibited by the interaction with certain proteins, and its expression can be controlled. we report here a novel aspect of mal gene regulation: the effect of cytoplasmic glucose and glucokinase (glk) on the activity and the expression of malt. amylomaltase (malq) is essential for the metabolism of maltose. it forms maltodextrins and glucose from mal ...200919028900
biochemical and structural properties of mouse kynurenine aminotransferase iii.kynurenine aminotransferase iii (kat iii) has been considered to be involved in the production of mammalian brain kynurenic acid (kyna), which plays an important role in protecting neurons from overstimulation by excitatory neurotransmitters. the enzyme was identified based on its high sequence identity with mammalian kat i, but its activity toward kynurenine and its structural characteristics have not been established. in this study, the biochemical and structural properties of mouse kat iii (m ...200919029248
biochemical and structural properties of mouse kynurenine aminotransferase iii.kynurenine aminotransferase iii (kat iii) has been considered to be involved in the production of mammalian brain kynurenic acid (kyna), which plays an important role in protecting neurons from overstimulation by excitatory neurotransmitters. the enzyme was identified based on its high sequence identity with mammalian kat i, but its activity toward kynurenine and its structural characteristics have not been established. in this study, the biochemical and structural properties of mouse kat iii (m ...200919029248
rrna suppressor of a eukaryotic translation initiation factor 5b/initiation factor 2 mutant reveals a binding site for translational gtpases on the small ribosomal subunit.the translational gtpases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. mutations that impair gtp hydrolysis by eukaryotic translation initiation factor 5b/initiation factor 2 (eif5b/if2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. a mutation in helix h5 of the 18s rrna in the 40s ribosomal subunit and intragenic mutations in domain ii of eif5b suppress the toxic effects associated wit ...200919029250
rrna suppressor of a eukaryotic translation initiation factor 5b/initiation factor 2 mutant reveals a binding site for translational gtpases on the small ribosomal subunit.the translational gtpases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. mutations that impair gtp hydrolysis by eukaryotic translation initiation factor 5b/initiation factor 2 (eif5b/if2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. a mutation in helix h5 of the 18s rrna in the 40s ribosomal subunit and intragenic mutations in domain ii of eif5b suppress the toxic effects associated wit ...200919029250
the chemistry and biochemistry of heme c: functional bases for covalent attachment.a discussion of the literature concerning the synthesis, function, and activity of heme c-containing proteins is presented. comparison of the properties of heme c, which is covalently bound to protein, is made to heme b, which is bound noncovalently. a question of interest is why nature uses biochemically expensive heme c in many proteins when its properties are expected to be similar to heme b. considering the effects of covalent heme attachment on heme conformation and on the proximal histidin ...200819030605
function and ribosomal localization of aif6, a translational regulator shared by archaea and eukarya.the translation factor if6 is shared by the archaea and the eukarya, but is not found in bacteria. the properties of eukaryal if6 (eif6) have been extensively studied, but remain somewhat elusive. eif6 behaves as a ribosome-anti-association factor and is involved in mirna-mediated gene silencing; however, it also seems to participate in ribosome synthesis and export. here we have determined the function and ribosomal localization of the archaeal (sulfolobus solfataricus) if6 homologue (aif6). we ...200919036786
function and ribosomal localization of aif6, a translational regulator shared by archaea and eukarya.the translation factor if6 is shared by the archaea and the eukarya, but is not found in bacteria. the properties of eukaryal if6 (eif6) have been extensively studied, but remain somewhat elusive. eif6 behaves as a ribosome-anti-association factor and is involved in mirna-mediated gene silencing; however, it also seems to participate in ribosome synthesis and export. here we have determined the function and ribosomal localization of the archaeal (sulfolobus solfataricus) if6 homologue (aif6). we ...200919036786
the ua_handle: a versatile submotif in stable rna architectures.stable rnas are modular and hierarchical 3d architectures taking advantage of recurrent structural motifs to form extensive non-covalent tertiary interactions. sequence and atomic structure analysis has revealed a novel submotif involving a minimal set of five nucleotides, termed the ua_handle motif (5'xu/an(n)x3'). it consists of a u:a watson-crick: hoogsteen trans base pair stacked over a classic watson-crick base pair, and a bulge of one or more nucleotides that can act as a handle for making ...200819036788
the ua_handle: a versatile submotif in stable rna architectures.stable rnas are modular and hierarchical 3d architectures taking advantage of recurrent structural motifs to form extensive non-covalent tertiary interactions. sequence and atomic structure analysis has revealed a novel submotif involving a minimal set of five nucleotides, termed the ua_handle motif (5'xu/an(n)x3'). it consists of a u:a watson-crick: hoogsteen trans base pair stacked over a classic watson-crick base pair, and a bulge of one or more nucleotides that can act as a handle for making ...200819036788
minor changes largely restore catalytic activity of archaeal rnase p rna from methanothermobacter thermoautotrophicus.the increased protein proportion of archaeal and eukaryal ribonuclease (rnase) p holoenzymes parallels a vast decrease in the catalytic activity of their rna subunits (p rnas) alone. we show that a few mutations toward the bacterial p rna consensus substantially activate the catalytic (c-) domain of archaeal p rna from methanothermobacter, in the absence and presence of the bacterial rnase p protein. large increases in ribozyme activity required the cooperative effect of at least two structural ...200919036794
minor changes largely restore catalytic activity of archaeal rnase p rna from methanothermobacter thermoautotrophicus.the increased protein proportion of archaeal and eukaryal ribonuclease (rnase) p holoenzymes parallels a vast decrease in the catalytic activity of their rna subunits (p rnas) alone. we show that a few mutations toward the bacterial p rna consensus substantially activate the catalytic (c-) domain of archaeal p rna from methanothermobacter, in the absence and presence of the bacterial rnase p protein. large increases in ribozyme activity required the cooperative effect of at least two structural ...200919036794
common thiolation mechanism in the biosynthesis of trna thiouridine and sulphur-containing cofactors.2-thioribothymidine (s(2)t), a modified uridine, is found at position 54 in transfer rnas (trnas) from several thermophiles; s(2)t stabilizes the l-shaped structure of trna and is essential for growth at higher temperatures. here, we identified an atpase (trna-two-thiouridine c, ttuc) required for the 2-thiolation of s(2)t in thermus thermophilus and examined in vitro s(2)t formation by ttuc and previously identified s(2)t-biosynthetic proteins (ttua, ttub, and cysteine desulphurases). the c-ter ...200819037260
structure and non-essential function of glycerol kinase in plasmodium falciparum blood stages.malaria pathology is caused by multiplication of asexual parasites within erythrocytes, whereas mosquito transmission of malaria is mediated by sexual precursor cells (gametocytes). microarray analysis identified glycerol kinase (gk) as the second most highly upregulated gene in plasmodium falciparum gametocytes with no expression detectable in asexual blood stage parasites. phosphorylation of glycerol by gk is the rate-limiting step in glycerol utilization. deletion of this gene from p. falcipa ...200919040641
urm1 at the crossroad of modifications. 'protein modifications: beyond the usual suspects' review series.the ubiquitin-like protein urm1 can be covalently conjugated to other proteins, such as the yeast thioredoxin peroxidase protein ahp1p, through a mechanism involving the ubiquitin e1-like enzyme uba4. recent findings have revealed a second function of urm1 as a sulphur carrier in the thiolation of eukaryotic cytoplasmic transfer rnas (trnas). interestingly, this new role of urm1 is similar to the sulphur-carrier activity of its prokaryotic counterparts, strengthening the hypothesis that urm1 is ...200819047990
a novel dimerization motif in the c-terminal domain of the thermus thermophilus dead box helicase hera confers substantial flexibility.dead box helicases are involved in nearly all aspects of rna metabolism. they share a common helicase core, and may comprise additional domains that contribute to rna binding. the thermus thermophilus helicase hera is the first dimeric dead box helicase. crystal structures of hera fragments reveal a bipartite c-terminal domain with a novel dimerization motif and an rna-binding module. we provide a first glimpse on the additional rna-binding module outside the hera helicase core. the dimerization ...200919050012
purification, crystallization and x-ray structures of the two manganese superoxide dismutases from caenorhabditis elegans.caenorhabditis elegans expresses two manganese superoxide dismutase enzymes (mnsod-2 and mnsod-3) that are targeted to the mitochondrion. mnsod-2 is constitutively expressed, while synthesis of mnsod-3 is inducible. the structures of these two mononuclear metalloenzymes have been determined to 1.8 and 1.7 a resolution, respectively. pink crystals formed in space group p4(1)2(1)2 for each, with unit-cell parameters a = b = 81.0, c = 137.4 a for mnsod-2 and a = b = 81.8, c = 136.0 a for mnsod-3. t ...200819052361
cloning, expression, crystallization and preliminary x-ray crystallographic analysis of 3-dehydroquinate synthase, xoo1243, from xanthomonas oryzae pv. oryzae.the disease bacterial blight results in serious production losses of rice in asian countries. the arob gene encoding dehydroquinate synthase (dhqs), which is a potential antibiotic target, was identified from the plant-pathogenic bacterium xanthomonas oryzae pv. oryzae (xoo). dhqs plays an essential role in the synthesis of aromatic compounds in the shikimate pathway. the arob gene (xoo1243) was cloned from xoo and the corresponding dhqs protein was subsequently overexpressed in escherichia coli ...200819052366
crystallization and preliminary x-ray diffraction studies of the prototypal homologue of mitoneet (tth-neet0026) from the extreme thermophile thermus thermophilus hb8.mitoneet (a mammalian mitochondrial outer membrane protein) is a potential pharmacological and clinical target of the insulin-sensitizer pioglitazone. the thermophilic homologue of mitoneet (ttha0026) from thermus thermophilus hb8 has been heterologously overproduced in escherichia coli and purified as a water-soluble prototypal protein containing the mitoneet-like [2fe-2s] cluster. the resultant recombinant protein, named tth-neet0026, has been crystallized in its oxidized form by the hanging-d ...200819052371
the 1.6 a crystal structure of mycobacterium smegmatis mshc: the penultimate enzyme in the mycothiol biosynthetic pathway.mycobacterium smegmatis mshc catalyzes the atp-dependent condensation of glcn-ins and l-cysteine to form l-cys-glcn-ins, the penultimate step in mycothiol biosynthesis. attempts to crystallize the native, full-length mshc have been unsuccessful. however, incubation of the enzyme with the cysteinyl adenylate analogue, 5'-o-[n-(l-cysteinyl)-sulfamonyl]adenosine (csa), followed by a 24-h limited trypsin proteolysis yielded an enzyme preparation that readily crystallized. the three-dimensional struc ...200819053270
classification and regression tree (cart) analyses of genomic signatures reveal sets of tetramers that discriminate temperature optima of archaea and bacteria.classification and regression tree (cart) analysis was applied to genome-wide tetranucleotide frequencies (genomic signatures) of 195 archaea and bacteria. although genomic signatures have typically been used to classify evolutionary divergence, in this study, convergent evolution was the focus. temperature optima for most of the organisms examined could be distinguished by cart analyses of tetranucleotide frequencies. this suggests that pervasive (nonlinear) qualities of genomes may reflect cer ...200719054742
classification and regression tree (cart) analyses of genomic signatures reveal sets of tetramers that discriminate temperature optima of archaea and bacteria.classification and regression tree (cart) analysis was applied to genome-wide tetranucleotide frequencies (genomic signatures) of 195 archaea and bacteria. although genomic signatures have typically been used to classify evolutionary divergence, in this study, convergent evolution was the focus. temperature optima for most of the organisms examined could be distinguished by cart analyses of tetranucleotide frequencies. this suggests that pervasive (nonlinear) qualities of genomes may reflect cer ...200719054742
recombinant production and biochemical characterization of a hyperthermostable alpha-glucan/maltodextrin phosphorylase from pyrococcus furiosus.alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-d-glucose-1-phosphate. we report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (pf1535) from a hyperthermophilic archaeon, pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. the apparent 98 kda recombinant enzyme was active over a broad range of temperatures and ...200819054743
recombinant production and biochemical characterization of a hyperthermostable alpha-glucan/maltodextrin phosphorylase from pyrococcus furiosus.alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-d-glucose-1-phosphate. we report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (pf1535) from a hyperthermophilic archaeon, pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. the apparent 98 kda recombinant enzyme was active over a broad range of temperatures and ...200819054743
identification of a new endogenous metabolite and the characterization of its protein interactions through an immobilization approach.the emerging field of global mass-based metabolomics provides a platform for discovering unknown metabolites and their specific biochemical pathways. we report the identification of a new endogenous metabolite, n(4)-(n-acetylaminopropyl)spermidine and the use of a novel proteomics based method for the investigation of its protein interaction using metabolite immobilization on agarose beads. the metabolite was isolated from the organism pyrococcus furiosus, and structurally characterized through ...200919055353
bridge helix and trigger loop perturbations generate superactive rna polymerases.cellular rna polymerases are highly conserved enzymes that undergo complex conformational changes to coordinate the processing of nucleic acid substrates through the active site. two domains in particular, the bridge helix and the trigger loop, play a key role in this mechanism by adopting different conformations at various stages of the nucleotide addition cycle. the functional relevance of these structural changes has been difficult to assess from the relatively small number of static crystal ...200819055851
differential effects of mitochondrial complex i inhibitors on production of reactive oxygen species.we have investigated the production of reactive oxygen species (ros) by complex i in isolated open bovine heart submitochondrial membrane fragments during forward electron transfer in presence of nadh, by means of the probe 2',7'-dichlorodihydrofluorescein diacetate. ros production by complex i is strictly related to its inhibited state. our results indicate that different complex i inhibitors can be grouped into two classes: class a inhibitors (rotenone, piericidin a and rolliniastatin 1 and 2) ...200919059197
differential effects of mitochondrial complex i inhibitors on production of reactive oxygen species.we have investigated the production of reactive oxygen species (ros) by complex i in isolated open bovine heart submitochondrial membrane fragments during forward electron transfer in presence of nadh, by means of the probe 2',7'-dichlorodihydrofluorescein diacetate. ros production by complex i is strictly related to its inhibited state. our results indicate that different complex i inhibitors can be grouped into two classes: class a inhibitors (rotenone, piericidin a and rolliniastatin 1 and 2) ...200919059197
how mitochondria produce reactive oxygen species.the production of ros (reactive oxygen species) by mammalian mitochondria is important because it underlies oxidative damage in many pathologies and contributes to retrograde redox signalling from the organelle to the cytosol and nucleus. superoxide (o2(*-)) is the proximal mitochondrial ros, and in the present review i outline the principles that govern o2(*-) production within the matrix of mammalian mitochondria. the flux of o2(*-) is related to the concentration of potential electron donors, ...200819061483
how mitochondria produce reactive oxygen species.the production of ros (reactive oxygen species) by mammalian mitochondria is important because it underlies oxidative damage in many pathologies and contributes to retrograde redox signalling from the organelle to the cytosol and nucleus. superoxide (o2(*-)) is the proximal mitochondrial ros, and in the present review i outline the principles that govern o2(*-) production within the matrix of mammalian mitochondria. the flux of o2(*-) is related to the concentration of potential electron donors, ...200819061483
superoxide dismutase from the eukaryotic thermophile alvinella pompejana: structures, stability, mechanism, and insights into amyotrophic lateral sclerosis.prokaryotic thermophiles supply stable human protein homologs for structural biology; yet, eukaryotic thermophiles would provide more similar macromolecules plus those missing in microbes. alvinella pompejana is a deep-sea hydrothermal-vent worm that has been found in temperatures averaging as high as 68 degrees c, with spikes up to 84 degrees c. here, we used cu,zn superoxide dismutase (sod) to test if this eukaryotic thermophile can provide insights into macromolecular mechanisms and stability ...200919063897
superoxide dismutase from the eukaryotic thermophile alvinella pompejana: structures, stability, mechanism, and insights into amyotrophic lateral sclerosis.prokaryotic thermophiles supply stable human protein homologs for structural biology; yet, eukaryotic thermophiles would provide more similar macromolecules plus those missing in microbes. alvinella pompejana is a deep-sea hydrothermal-vent worm that has been found in temperatures averaging as high as 68 degrees c, with spikes up to 84 degrees c. here, we used cu,zn superoxide dismutase (sod) to test if this eukaryotic thermophile can provide insights into macromolecular mechanisms and stability ...200919063897
crystal structure of a translation termination complex formed with release factor rf2.we report the crystal structure of a translation termination complex formed by the thermus thermophilus 70s ribosome bound with release factor rf2, in response to a uaa stop codon, solved at 3 a resolution. the backbone of helix alpha5 and the side chain of serine of the conserved spf motif of rf2 recognize u1 and a2 of the stop codon, respectively. a3 is unstacked from the first 2 bases, contacting thr-216 and val-203 of rf2 and stacking on g530 of 16s rrna. the structure of the rf2 complex sup ...200819064930
vectorial proton transfer coupled to reduction of o2 and no by a heme-copper oxidase.the heme-copper oxidase (hcuo) superfamily consists of integral membrane proteins that catalyze the reduction of either oxygen or nitric oxide. the hcuos that reduce o(2) to h(2)o couple this reaction to the generation of a transmembrane proton gradient by using electrons and protons from opposite sides of the membrane and by pumping protons from inside the cell or organelle to the outside. the bacterial no-reductases (nor) reduce no to n(2)o (2no + 2e(-) + 2h(+) --> n(2)o + h(2)o), a reaction a ...200819074284
structure and functional role of dynein's microtubule-binding domain.dynein motors move various cargos along microtubules within the cytoplasm and power the beating of cilia and flagella. an unusual feature of dynein is that its microtubule-binding domain (mtbd) is separated from its ring-shaped aaa+ adenosine triphosphatase (atpase) domain by a 15-nanometer coiled-coil stalk. we report the crystal structure of the mouse cytoplasmic dynein mtbd and a portion of the coiled coil, which supports a mechanism by which the atpase domain and mtbd may communicate through ...200819074350
upf201 archaeal specific family members reveal structural similarity to rna-binding proteins but low likelihood for rna-binding function.we have determined x-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (upf0201; pfam classification: duf54) to advance our understanding of the genetic repertoire of archaea. despite low pairwise amino acid sequence identities (10-40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. their common polypeptide chain fold, encompassing a five-stranded antiparal ...200819079550
heterogeneity of large macromolecular complexes revealed by 3d cryo-em variance analysis.macromolecular structure determination by cryo-electron microscopy (em) and single-particle analysis are based on the assumption that imaged molecules have identical structure. with the increased size of processed data sets, it becomes apparent that many complexes coexist in a mixture of conformational states or contain flexible regions. we describe an implementation of the bootstrap resampling technique that yields estimates of voxel-by-voxel variance of a structure reconstructed from the set o ...200819081053
structural basis for inactivation of the human pyruvate dehydrogenase complex by phosphorylation: role of disordered phosphorylation loops.we report the crystal structures of the phosporylated pyruvate dehydrogenase (e1p) component of the human pyruvate dehydrogenase complex (pdc). the complete phosphorylation at ser264-alpha (site 1) of a variant e1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the pdc core. we show that unlike its unmodified counterpart, the presence of a phosphoryl group at ser264-alpha prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three ph ...200819081061
effect of simulated microgravity on oxidation-sensitive gene expression in pc12 cells.oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). a baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. the objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (pc12)] under simulated micro-g conditions. the pc12 cell line is well cha ...200619081771
large deviations for random trees and the branching of rna secondary structures.we give a large deviation principle (ldp) with explicit rate function for the distribution of vertex degrees in plane trees, a combinatorial model of rna secondary structures. we calculate the typical degree distributions based on nearest neighbor free energies, and compare our results with the branching configurations found in two sets of large rna secondary structures. we find substantial agreement overall, with some interesting deviations which merit further study.200819083065
large deviations for random trees and the branching of rna secondary structures.we give a large deviation principle (ldp) with explicit rate function for the distribution of vertex degrees in plane trees, a combinatorial model of rna secondary structures. we calculate the typical degree distributions based on nearest neighbor free energies, and compare our results with the branching configurations found in two sets of large rna secondary structures. we find substantial agreement overall, with some interesting deviations which merit further study.200819083065
a bridge to transcription by rna polymerase.a comprehensive survey of single amino-acid substitution mutations critical for rna polymerase function published in journal of biology supports a proposed mechanism for polymerase action in which movement of the polymerase 'bridge helix' promotes transcriptional activity in cooperation with a critical substrate-interaction domain, the 'trigger loop'.200819090964
structure of an argonaute silencing complex with a seed-containing guide dna and target rna duplex.here we report on a 3.0 a crystal structure of a ternary complex of wild-type thermus thermophilus argonaute bound to a 5'-phosphorylated 21-nucleotide guide dna and a 20-nucleotide target rna containing cleavage-preventing mismatches at the 10-11 step. the seed segment (positions 2 to 8) adopts an a-helical-like watson-crick paired duplex, with both ends of the guide strand anchored in the complex. an arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in a ...200819092929
a signal relay between ribosomal protein s12 and elongation factor ef-tu during decoding of mrna.codon recognition by aminoacyl-trna on the ribosome triggers a process leading to gtp hydrolysis by elongation factor tu (ef-tu) and release of aminoacyl-trna into the a site of the ribosome. the nature of this signal is largely unknown. here, we present genetic evidence that a specific set of direct interactions between ribosomal protein s12 and aminoacyl-trna, together with contacts between s12 and 16s rrna, provide a pathway for the signaling of codon recognition to ef-tu. three novel amino a ...200919095621
mitochondrial dna damage in iron overload.chronic iron overload has slow and insidious effects on heart, liver, and other organs. because iron-driven oxidation of most biologic materials (such as lipids and proteins) is readily repaired, this slow progression of organ damage implies some kind of biological "memory." we hypothesized that cumulative iron-catalyzed oxidant damage to mtdna might occur in iron overload, perhaps explaining the often lethal cardiac dysfunction. real time pcr was used to examine the "intactness" of mttdna in cu ...200919095657
functional role of coenzyme q in the energy coupling of nadh-coq oxidoreductase (complex i): stabilization of the semiquinone state with the application of inside-positive membrane potential to proteoliposomes.coenzyme q10 (which is also designated as coq10, ubiquinone-10, uq10, coq, uq or simply as q) plays an important role in energy metabolism. for nadh-q oxidoreductase (complex i), ohnishi and salerno proposed a hypothesis that the proton pump is operated by the redox-driven conformational change of a q-binding protein, and that the bound form of semiquinone (sq) serves as its gate [febs letters 579 (2005) 45-55]. this was based on the following experimental results: (i) epr signals of the fast-re ...200819096096
functional specialization of transcription elongation factors.elongation factors nusg and rfah evolved from a common ancestor and utilize the same binding site on rna polymerase (rnap) to modulate transcription. however, although nusg associates with rnap transcribing most escherichia coli genes, rfah regulates just a few operons containing ops, a dna sequence that mediates rfah recruitment. here, we describe the mechanism by which this specificity is maintained. we observe that rfah action is indeed restricted to those several operons that are devoid of n ...200919096362
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