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PubMed1976

On the kinetics of the rifampicin-RNA-polymerase complex. Differences between crude and purified enzyme fractions.

The antibiotic rifampicin forms a very tight complex with DNA-dependent RNA polymerase of Escherichia coli. The rate constants of association and dissociation of this complex have been measured and found to be dependent on the purity of the enzyme. Thus a crude RNA polymerase (fraction-3 enzyme) has rate constants different from those of an enzyme further purified by DEAE-cellulose chromatography (fraction-4 enzyme). The complex produced by the antibiotic and the fraction-3 enzyme is about ten times more stable and is formed about ten times more slowly than the complex with fraction-4 enzyme. It has been shown that the RNA present in the crude enzyme and removed by chromatography on DEAE-cellulose is the cause of the change in the kinetics of the complex. tRNA of rat liver and crude rat liver RNA added to purified RNA polymerase have a similar effect. Mg2+, which has no intrinsic influence, augments the effect of the nucleic acids, whereas monovalent cations do not. Since nucleic acids increase the stability of the complex, but at the same time decrease the rat of its formation, the equilibrium constant, Keq, remains almost the same. The possible effects of nucleic acids on the rifampicin binding site are discussed.

Ammonium Sulfate↗
PubMed2006

The novel A4435G mutation in the mitochondrial tRNAMet may modulate the phenotypic expression of the LHON-associated ND4 G11778A mutation.

PURPOSE: To investigating the role of mitochondrial haplotypes in the development of Leber's hereditary optic neuropathy (LHON) associated with the ND4 G11778A mutation in Chinese families. METHODS: A three-generation Chinese family with LHON was studied by clinical and genetic evaluation as well as molecular and biochemical analysis of mitochondrial (mt)DNA. RESULTS: This family exhibits a high penetrance and expressivity of visual impairment. The average age at onset was 13.9 years in this family. Of the family members, 86% of the male and 29% of the female matrilineal relatives had visual loss, with a wide range of severity, from blindness to nearly normal vision. Molecular analysis of mtDNA identified the homoplasmic ND4 G11778A mutation and 35 other variants, belonging to the Asian haplogroup D5. Of other variants, the novel homoplasmic A4435G mutation absent in 164 Chinese controls is localized at 3' end adjacent to the anticodon, at conventional position 37 (A37), of tRNAMet. The adenine (A37) at this position of tRNAMet is extraordinarily conserved from bacteria to human mitochondria. This modified A37 was shown to contribute to the high fidelity of codon recognition and to the structural formation and stabilization of functional tRNAs. In fact, the significant reduction of the steady state levels in tRNAMet was observed in cells carrying the both the A4435G and G11778A mutations but not cells carrying only the G11778A mutation. Thus, a failure in mitochondrial tRNA metabolism, caused by the A4435G mutation, may worsen the mitochondrial dysfunction associated with the primary G11778A mutation. CONCLUSIONS: The novel tRNAMet A4435G mutation has a potential modifier role in increasing the penetrance and expressivity of the primary LHON-associated G11778A mutation in this Chinese family.

Adolescent↗
PubMed1977

[Primary structure of functions of RNA terminals].

Primary structure of tRNA, rRNA, nRNA, mRNA and viral RNA terminals has been studied. Various RNAs responsible for similar functions have been shown to possess some common features. Previously obtained data on the functional role of RNA terminal sequences, mechanisms of their biosynthesis, the stabilizing role of 5'- and 3'-sequences for the molecules of translated eukaryotic RNAs and some viral RNAs are reviewed.

Base Sequence↗
PubMed2000

Active site of lysyl-tRNA synthetase: structural studies of the adenylation reaction.

Aminoacyl-tRNA synthetases play a key role in protein biosynthesis by catalyzing the specific aminoacylation of tRNA. The energy required for the formation of the ester bond between the amino acid carboxylate group and the tRNA acceptor stem is supplied by coupling the reaction to the hydrolysis of ATP. Lysyl-tRNA synthetase from Escherichia coli belongs to the family of class II synthetases and carries out a two-step reaction, in which lysine is activated by being attached to the alpha-phosphate of AMP before being transferred to the cognate tRNA. Crystals of the thermo-inducible E. coli lysyl-tRNA synthetase LysU which diffract to 2.1 A resolution have been used to determine crystal structures of the enzyme in the presence of lysine, the lysyl-adenylate intermediate, and the nonhydrolyzable ATP analogue AMP-PCP. Additional data have been obtained from crystals soaked in a solution containing ATP and Mn(2+). The refined crystal structures give "snapshots" of the active site corresponding to key steps in the aminoacylation reaction and provide the structural framework for understanding the mechanism of lysine activation. The active site of LysU is shaped to position the substrates for the nucleophilic attack of the lysine carboxylate on the ATP alpha-phosphate. No residues are directly involved in catalysis, but a number of highly conserved amino acids and three metal ions coordinate the substrates and stabilize the pentavalent transition state. A loop close to the catalytic pocket, disordered in the lysine-bound structure, becomes ordered upon adenine binding.

Adenosine Triphosphate↗
PubMed1998

[Two cases of polymyositis associated with interstitial pneumonia with anti-OJ (isoleucyl tRNA synthetase) antibodies].

We present two cases of polymyositis (PM) associated with interstitial pneumonia (IP) whose sera contain autoantibodies to OJ (isoleucyl tRNA synthetase). The first patient is a 51 year-old female who was diagnosed as rheumatoid arthritis (RA) and treated with gold and corticosteroid at another hospital. She was admitted to Keio University Hospital due to worsening of dyspnea on exertion and polyarthritis. Laboratory findings revealed elevation of serum CK and LDH. A diagnosis of PM was made based on the myogenic pattern of EMG and pathological feature by muscle biopsy. Chest radiography and CT showed interstitial fibrosis. Because of clinical deterioration, the dose of corticosteroid was increased (prednisolone 50 mg/day) and her symptom was stabilized. The second patient, a 62 year-old male, was admitted to Kawasaki Municipal Hospital because of dyspnea on exertion, polyarthritis, and fever. He was diagnosed as PM associated with IP on the basis of his clinical and laboratory findings, and chest radiography. He was treated with methylprednisolone pulse therapy (800 mg/day for three days) and his symptoms were improved. Both patients were found to have autoantibodies to OJ. Autoantibodies to aminoacyl tRNA synthetase have been described to be associated with myositis and/or IP. In North American, it was reported that all patients with anti-OJ had either myositis or IP or both. This suggests that anti-OJ was commonly associated with the anti-synthetase syndrome observed with other anti-synthetases. This is the first report of Japanese patients with anti-OJ antibody. The clinical features of these patients were likely to be similar to those observed in North American patients. However, further studies are necessary to clarify the precise clinical significance of this antibody.

Autoantibodies↗
PubMed1996

Interstitial lung disease with autoantibodies against aminoacyl-tRNA synthetases in the absence of clinically apparent myositis.

Autoantibodies against aminoacyl-tRNA synthetases (antisynthetases) have been found to be highly specific for polymyositis and dermatomyositis and to correlate strongly with complicating interstitial lung disease (ILD). We describe the clinical presentations and course of 10 patients with ILD and anti-synthetase antibodies in whom underlying myositis was not clinically evident. Anti-PL-12 antibodies (antialanyl-tRNA synthetase) were most common (60%), followed by anti-Jo-1 (antihistidyl-tRNA synthetase) and anti-OJ (anti-isoleucyl-tRNA synthetase) (20% each). All 10 patients had anticytoplasmic antibodies by indirect immunofluorescence on HEp-2 cells. Five of 10 presented with features of connective tissue disease, whereas two presented with acute respiratory failure, two with insidious onset of diminished exercise tolerance, and one with persistent cough. All but one patient received corticosteroids, four were given oral cyclophosphamide, and two azathioprine. ILD resolved or stabilized in five patients (50%), and progressed in four (40%). The "antisynthetase syndrome" may occur in the absence of clinical myositis, and the ILD in these patients is usually responsive to therapy. Antisynthetase testing should be considered in patients with ILD who have a cytoplasmic pattern by antinuclear antibody (ANA) testing on HEp-2 cells, because early recognition and treatment of such patients affects their clinical course.

Adult↗
PubMed2006

Computational studies of tryptophanyl-tRNA synthetase: activation of ATP by induced-fit.

Catalysis of amino acid activation by Bacillus stearothermophilus tryptophanyl-tRNA synthetase involves three allosteric states: (1) Open; (2) closed pre-transition state (PreTS); and (3) closed products (Product). The interconversions of these states entail significant domain motions driven by ligand binding. We explore the application of molecular dynamics simulations to investigate ligand-linked conformational stability changes associated with this catalytic cycle. Multiple molecular dynamics trajectories (5 ns) for 11 distinct liganded and unliganded monomer configurations show that the PreTS conformation is unstable in the absence of ATP, reverting within approximately 600 ps nearly to the Open conformation. In contrast, Open and Product state trajectories were stable, even without ligands, confirming the previous suggestion that catalysis entails destabilization of the protein conformation, driven by ATP-binding energies developed as the PreTS state assembles during induced-fit. The simulations suggest novel mechanistic details associated with both induced-fit (Open-PreTS) and catalysis (PreTS-Product). Notably, Mg2+ -ATP interactions are coupled to interactions between ATP and active-site lysine side-chains via mechanisms that cannot be captured under the molecular mechanics approximations, and which therefore require restraining potentials for stable simulation. Simulations of Mg2+. ATP-bound PreTS complexes with restraining potentials and with a virtual K111A mutant confirm that these coupling interactions are necessary to sustain the PreTS conformation and, in turn, provide a new model for how the PreTS conformation activates ATP for catalysis. These results emphasize the central role of the PreTS state as a high-energy intermediate structure along the catalytic pathway and suggest that Mg2+ and the KMSKS loop function cooperatively during catalysis.

Adenosine Triphosphate↗
PubMed1986

Affinities of tRNA binding sites of ribosomes from Escherichia coli.

The binding affinities of tRNAPhe, Phe-tRNAPhe, and N-AcPhe-tRNAPhe from either Escherichia coli or yeast to the P, A, and E sites of E. coli 70S ribosomes were determined at various ionic conditions. For the titrations, both equilibrium (fluorescence) and nonequilibrium (filtration) techniques were used. Site-specific rather than stoichiometric binding constants were determined by taking advantage of the varying affinities, stabilities, and specificities of the three binding sites. The P site of poly(U)-programmed ribosomes binds tRNAPhe and N-AcPhe-tRNAPhe with binding constants in the range of 10(8) M-1 and 5 X 10(9) M-1, respectively. Binding to the A site is 10-200 times weaker, depending on the Mg2+ concentration. Phe-tRNAPhe binds to the A site with a similar affinity. Coupling A site binding of Phe-tRNAPhe to GTP hydrolysis, by the addition of elongation factor Tu and GTP, leads to an apparent increase of the equilibrium constant by at least a factor of 10(4). Upon omission of poly(U), the affinity of the P site is lowered by 2-4 orders of magnitude, depending on the ionic conditions, while A site binding is not detectable anymore. The affinity of the E site, which specifically binds deacylated tRNAPhe, is comparable to that of the A site. In contrast to P and A sites, binding to the E site is labile and insensitive to changes of the ionic strength. Omission of the mRNA lowers the affinity at most by a factor of 4, suggesting that there is no efficient codon-anticodon interaction in the E site. On the basis of the equilibrium constants, the displacement step of translocation, to be exergonic, requires that the tRNA leaving the P site is bound to the E site. Under in vivo conditions, the functional role of transient binding of the leaving tRNA to the E site, or a related site, most likely is to enhance the rate of translocation.

Binding Sites↗
PubMed2003

Crystal structure of the transfer-RNA domain of transfer-messenger RNA in complex with SmpB.

Accurate translation of genetic information into protein sequence depends on complete messenger RNA molecules. Truncated mRNAs cause synthesis of defective proteins, and arrest ribosomes at the end of their incomplete message. In bacteria, a hybrid RNA molecule that combines the functions of both transfer and messenger RNAs (called tmRNA) rescues stalled ribosomes, and targets aberrant, partially synthesized, proteins for proteolytic degradation. Here we report the 3.2-A-resolution structure of the tRNA-like domain of tmRNA (tmRNA(Delta)) in complex with small protein B (SmpB), a protein essential for biological functions of tmRNA. We find that the flexible RNA molecule adopts an open L-shaped conformation and SmpB binds to its elbow region, stabilizing the single-stranded D-loop in an extended conformation. The most striking feature of the structure of tmRNA(Delta) is a 90 degrees rotation of the TPsiC-arm around the helical axis. Owing to this unusual conformation, the SmpB-tmRNA(Delta) complex positioned into the A-site of the ribosome orients SmpB towards the small ribosomal subunit, and directs tmRNA towards the elongation-factor binding region of the ribosome. On the basis of this structure, we propose a model for the binding of tmRNA on the ribosome.

Bacterial Proteins↗
PubMed1997

Mitogenic factor secreted by Streptococcus pyogenes is a heat-stable nuclease requiring His122 for activity.

The gene encoding a mitogenic factor, termed MF, was cloned from Streptococcus pyogenes and the recombinant MF was overexpressed in Escherichia coli. Both the natural and recombinant MF had heat-resistant nuclease activity. The nuclease activity of MF was characterized using the recombinant protein. MF showed endonuclease activity, digesting ssDNA, dsDNA and tRNA. The optimal pH for the DNase activity of MF was 9.5. The DNase activity was enhanced approximately tenfold by the simultaneous presence of two divalent cations, Mg2+ and Ca2+, compared to either alone and was inhibited by EDTA or NaCl. The heat stability of MF was biphasic; the DNase activity was heat-stable from 0 to 50 degrees C over 80 degrees C but very unstable at around 60 degrees C. DNA digested by MF possessed 5'-phosphorylated and 3'-hydroxylated termini, identical to those obtained by digestion of DNA by pancreatic deoxyribonuclease I. A mutant clone revealed that His122 was a residue essential to the nuclease activity.

Amino Acid Sequence↗
PubMed2005

Plant 7SL RNA genes belong to type 4 of RNA polymerase III- dependent genes that are composed of mixed promoters.

The genes transcribed by RNA polymerase III (pol III) display a great diversity in terms of promoter structure and are placed in four groups accordingly. Type 3 subset of pol III genes has promoter elements which reside entirely upstream of the coding region of the gene whereas type 4 consists of genes with mixed promoters that enclose intra- and extragenic regulatory sequences. Plant 7SL RNA genes have been previously classified as type 3 of pol III genes requiring an upstream sequence element and a canonical TATA box for transcriptional activity in transfected plant protoplasts. We have identified two novel functional control regions within the coding region of an Arabidopsis 7SL RNA gene (At7SL-1) that resemble tRNA gene-specific A and B boxes with respect to sequence and position. Single and multiple nucleotide substitutions in either of these regions resulted in a pronounced reduction of transcription activity in tobacco nuclear extract that was not caused by a decreased stability as shown by decay kinetics of wild type and mutant RNA transcripts. These findings suggest that plant 7SL RNA genes should be actually placed in type 4 of pol III-transcribed genes. As a consequence of substantially different upstream promoters utilized by plant and human pol III, in vitro transcription of 7SL RNA genes in heterologous systems is severely impaired. A chimeric human 7SL RNA gene that contains the 5' flanking region up to position -300 of At7SL-1 is yet transcribed with a reduced efficiency in tobacco extract when compared with the plant wild-type gene, supporting the notion that internal regulatory elements contribute to full activity.

Arabidopsis↗
PubMed1995

Minor groove recognition of the conserved G.U pair at the Tetrahymena ribozyme reaction site.

The guanine-uracil (G.U) base pair that helps to define the 5'-splice site of group I introns is phylogenetically highly conserved. In such a wobble base pair, G makes two hydrogen bonds with U in a geometry shifted from that of a canonical Watson-Crick pair. The contribution made by individual functional groups of the G.U pair in the context of the Tetrahymena ribozyme was examined by replacement of the G.U pair with synthetic base pairs that maintain a wobble configuration, but that systematically alter functional groups in the major and minor grooves of the duplex. The substitutions demonstrate that the exocyclic amine of G, when presented on the minor groove surface by the wobble base pair conformation, contributes substantially (2 kilocalories.mole-1) to binding by making a tertiary interaction with the ribozyme active site. It contributes additionally to transition state stabilization. The ribozyme active site also makes tertiary contacts with a tripod of 2'-hydroxyls on the minor groove surface of the splice site helix. This suggests that the ribozyme binds the duplex primarily in the minor groove. The alanyl aminoacyl transfer RNA (tRNA) synthetase recognizes the exocyclic amine of an invariant G.U pair and contacts a similar array of 2'-hydroxyls when binding the tRNA(Ala) acceptor stem, providing an unanticipated parallel between protein-RNA and RNA-RNA interactions.

Animals↗
PubMed1996

A tyrosyl-tRNA synthetase suppresses structural defects in the two major helical domains of the group I intron catalytic core.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase, the CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. The group I intron catalytic core is thought to consist of two extended helical domains, one formed by coaxial stacking of P5, P4, P6, and P6a (P4-P6 domain) and the other consisting of P8, P3, P7, and P9 (P3-P9 domain). To investigate how CYT-18 stabilizes the active RNA structure, we used an Escherichia coli genetic assay based on the phage T4 td intron to systematically test the ability of CYT-18 to compensate for structural defects in three key regions of the catalytic core: J3/4 and J6/7, connecting regions that form parts of the triple-helical-scaffold structure with the P4-P6 domain, and P7, a long-range base-pairing interaction that forms the guanosine-binding site and is part of the P3-P9 domain. Our results show that CYT-18 can suppress numerous mutations that disrupt the J3/4 and J6/7 nucleotide-triple interactions, as well as mutations that disrupt base-pairing in P7. CYT-18 suppressed mutations of phylogenetically conserved nucleotide residues at all positions tested, except for the universally conserved G-residue at the guanosine-binding site. Structure mapping experiments with selected mutant introns showed that the CYT-18-suppressible J3/4 mutations primarily impaired folding of the P4-P6 domain, while the J6/7 mutations impaired folding of both the P4-P6 and P3-P9 domains to various degrees. The P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain. The finding that the P7 mutations also impaired formation of P3 provides evidence that the formation of these two long-range pairings is interdependent in the td intron. Considered together with previous work, the nature of mutations suppressed by CYT-18 supports a model in which CYT-18 helps assemble the P4-P6 domain and then stabilizes the two major helical domains of the catalytic core in the correct relative orientation to form the intron's active site.

Bacteriophage T4↗
PubMed1996

Disordered C-terminal domain of tyrosyl transfer-RNA synthetase: evidence for a folded state.

The C-terminal domain (residues 320 to 419) of tyrosyl-tRNA synthetase from Bacillus stearothermophilus (Bst-TyrRS) is necessary for the binding of tRNA(Tyr) but disordered in the crystal structure. Four different criteria showed that the isolated C-terminal domain of Bst-TyrRS was at least partially folded in solution. Its spectrum of circular dichroism was compatible with a high content of secondary structure elements (56% of its residues) and these structural elements disappeared in 7.5 M urea. It was unfolded by urea along a unique transition, around 6.0 M, according to the variations in the fluorescence of its tyrosine residues and in its electrophoretic mobility through transverse gradient gels. It was denatured by heat with a temperature of half-precipitation in 30 minutes that was equal to 67.9 degrees C and close to the Bst-TyrRS one, 68.7 degrees C. Its transitions of denaturation by urea or temperature were weakly cooperative. The C-terminal domains of the TyrRSs from Escherichia coli (Eco-TyrRS) and B. stearothermophilus could be genetically exchanged without a significant loss of aminoacylation activity. A hybrid between the N-terminal domain of Bst-TyrRS and the C-terminal domain of Eco-TyrRS was precipitated by heat in 30 minutes following two transitions: 83% of the molecules were precipitated with a temperature of half-transition (51.6 degrees C) close to the Eco-TyrRS one (48.6 degrees C). The remainder was precipitated with a temperature of half-transition (65.5 degrees C) close to the Bst-TyrRS one (67.2 degrees C) or that of its N-terminal domain (68.0 degrees C). These results showed that the C-terminal domain of Eco-TyrRS could undergo a transition from a soluble active conformation to an insoluble one. The denaturations of Bst-TyrRS and of its N-terminal domain by urea occurred with two successive transitions, around 4 M and 6 M, and thus according to a complex mechanism.

Base Sequence↗
PubMed2006

Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome.

The translocation of tRNA and mRNA through the ribosome is promoted by elongation factor G (EF-G), a GTPase that hydrolyzes GTP during the reaction. Recently, it was reported that, in contrast to previous observations, the affinity of EF-G was much weaker for GTP than for GDP and that ribosome-catalyzed GDP-GTP exchange would be required for translocation [Zavialov AV, Hauryliuk VV, Ehrenberg M (2005) J Biol 4:9]. We have reinvestigated GTP/GDP binding and show that EF-G binds GTP and GDP with affinities in the 20 to 40 microM range (37 degrees C), in accordance with earlier reports. Furthermore, GDP exchange, which is extremely rapid on unbound EF-G, is retarded, rather than accelerated, on the ribosome, which, therefore, is not a nucleotide-exchange factor for EF-G. The EF-G.GDPNP complex, which is very labile, is stabilized 30,000-fold by binding to the ribosome. These findings, together with earlier kinetic results, reveal that EF-G enters the pretranslocation ribosome in the GTP-bound form and indicate that, upon ribosome-complex formation, the nucleotide-binding pocket of EF-G is closed, presumably in conjunction with GTPase activation. GTP hydrolysis is required for rapid tRNA-mRNA movement, and P(i) release induces further rearrangements of both EF-G and the ribosome that are required for EF-G turnover.

Dissociative Disorders↗
PubMed2006

Molecular analysis of isoleucyl-tRNA synthetase mutations in clinical isolates of methicillin-resistant Staphylococcus aureus with low-level mupirocin resistance.

Emergence and spread of low-level mupirocin resistance in staphylococci have been increasingly reported in recent years. The aim of this study was to characterize missense mutations within the chromosomal isoleucyl-tRNA synthetase gene (ileS) among clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) with low-level mupirocin resistance. A total of 20 isolates of MRSA with low-level mupirocin resistance (minimal inhibitory concentration, 16-64 microg/mL) were collected from 79 patients in intensive care units for six months. The isolates were analyzed for isoleucyl-tRNA synthetase (IleS) mutations that might affect the binding of mupirocin to the three-dimensional structure of the S. aureus IleS enzyme. All isolates with low-level mupirocin resistance contained the known V588F mutation affecting the Rossman fold, and some of them additionally had previously unidentified mutations such as P187F, K226T, F227L, Q612H, or V767D. Interestingly, Q612H was a novel mutation that was involved in stabilizing the conformation of the catalytic loop containing the KMSKS motif. In conclusion, this study confirms that molecular heterogeneity in ileS gene is common among clinical MRSA isolates with low-level mupirocin resistance, and further study on clinical mutants is needed to understand the structural basis of low-level mupirocin resistance.

Drug Resistance, Bacterial↗