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PubMed1981

A small-angle X-ray scattering study of the complex formation between elongation factor Tu . GTP and valyl-tRNA Val I from Escherichia coli.

The complex formation between elongation factor Tu (EF-Tu) . GTP and valyl-tRNA Val 1 has been investigated using the small-angle X-ray scattering titration technique. The main species observed is a 1:1 complex with a stability constant log K greater than or equal to 6. The corresponding interaction between EF-Tu . GTP and non-aminoacylated tRNA appears to be much weaker with an estimated log K approximately equal to 4. The radius of gyration determined for the EF-Tu . GTP -- valyl-tRNA Val 1 complex is larger (R = 3.6 nm) than that of EF-Tu . GTP (R = 2.5 nm). Likewise, the maximum distance within this complex is larger (Dmax = 12.5 nm) than the one within EF-Tu . GTP (Dmax = 8.5 nm). These data as well as the p(r) curve are consistent with a multiellipsoid model for the complex. From this model it is indicated that the acceptor stem of tRNA is attached to EF-Tu and that the anticodon stem and loop protrude into the solution.

Bacterial Proteins↗
PubMed1980

Mechanism of translocation: effect of cognate transfer ribonucleic acids on the binding of AUGUn to 70S ribosomes.

We try to mimic the unidirectional sliding-type movement of the PP-tRNA . mRNA complex with respect to the ribosome by looking at the effect of different combinations of cognate tRNAs on the stability of the 70S-AUGUn complex. The association constant for the binary complex 70S-AUGU3 was determined as 6.8 x 10(5) M-1. Addition of tRNAfMet resulted in a 67-fold increase in the association constant, which with both cognate tRNAs is revised to Kassoc = 2.2 x 10(8) M-1. Increasing the chain length of the oligonucleotide from AUGU3 to AUGU13 did not further raise the association constant. The data indicate that the stability of the 70S ribosome . mRNA interaction is governed by the presence of the cognate tRNAs and is topographically restricted to the decoding domains. Since a peptidyl group in the tRNA increases the affinity of AUGU3 for the ribosome by up to 15-fold, we conclude that the affinity of the peptidyl transfer center for the peptidyl moiety pulls the PP-tRNA . mRNA complex from the A (aminoacyl-tRNA) site to the P (peptidyl-tRNA) site. EF-G . GTP or EF-G . GMPPCP 5'-(beta, gamma-methylene)triphosphate] displace tRNAfMet from the quaternary complex 70S . AUGUn . tRNAfMet . tRNAPhe (n = 3 and 6) at Mg2+ less than 25 mM. From the amount of EF-G . GTP bound to a 70S ribosome, it follows that the elongation factor replaces the deacylated tRNA in a stoichiometric way. These data indicate that the EF-G . GTP-dependent release of the deacylated tRNA from the P site, followed by removal of EF-G . GDP from the 50S subunit, is sufficient to trigger the translocation of the mRNA . PP-tRNA complex.

Biological Transport↗
PubMed2005

Complete translation of the hepatitis C virus genome in vitro: membranes play a critical role in the maturation of all virus proteins except for NS3.

We developed an in vitro translation extract from Krebs-2 cells that translates the entire open reading frame of the hepatitis C virus (HCV) strain H77 and properly processes the viral protein precursors when supplemented with canine microsomal membranes (CMMs). Translation of the C-terminal portion of the viral polyprotein in this system is documented by the synthesis of NS5B. Evidence for posttranslational modification of the viral proteins, the N-terminal glycosylation of E1 and the E2 precursor (E2-p7), and phosphorylation of NS5A is presented. With the exception of NS3, efficient generation of all virus-specific proteins is CMM dependent. A time course of the appearance of HCV products indicates that the viral polyprotein is cleaved cotranslationally. A competitive inhibitor of the NS3 protease inhibited accumulation of NS3, NS4B, NS5A, and NS5B, but not that of NS2 or structural proteins. CMMs also stabilized HCV mRNA during translation. Finally, the formyl-[35S]methionyl moiety of the initiator tRNA(Met) was incorporated exclusively into the core protein portion of the polyprotein, demonstrating that translation initiation in this system occurs with high fidelity.

Animals↗
PubMed2003

Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme.

Compact but non-native intermediates have been implicated in the hierarchical folding of several large RNAs, but there is little information on their structure. In this article, ribonuclease and hydroxyl radical cleavage protection assays showed that base pairing of core helices stabilize a compact state of a small group I ribozyme from Azoarcus pre-tRNA(ile). Base pairing of the ribozyme core requires 10-fold less Mg(2+) than stable tertiary interactions, indicating that assembly of helices in the catalytic core represents a distinct phase that precedes the formation of native tertiary structure. Tertiary folding occurs in <100 ms at 37 degrees C. Such rapid folding is unprecedented among group I ribozymes and illustrates the association between structural complexity and folding time. A 3D model of the Azoarcus ribozyme was constructed by identifying homologous sequence motifs in rRNA. The model reveals distinct structural features, such as a large interface between the P4-P6 and P3-P9 domains, that may explain the unusual stability of the Azoarcus ribozyme and the cooperativity of folding.

Azoarcus↗
PubMed2005

Two conserved threonines collaborate in the Escherichia coli leucyl-tRNA synthetase amino acid editing mechanism.

The aminoacyl-tRNA synthetases covalently link transfer RNAs to their cognate amino acids. Some of the tRNA synthetases have employed an editing mechanism to ensure fidelity in this first step of protein synthesis. The amino acid editing active site for Escherichia coli leucyl-tRNA synthetase resides within the CP1 domain that folds discretely from the main body of the enzyme. A portion of the editing active site is lined with conserved threonines. Previously, we identified one of these threonine residues (Thr(252)) as a critical amino acid specificity factor. On the basis of X-ray crystal structure information, two other nearby threonine residues (Thr(247) and Thr(248)) were hypothesized to interact with the editing substrate near its cleavage site. Single mutations of either of these conserved threonine residues had minimal effects on amino acid editing. However, double mutations that deleted the hydroxyl group from the neighboring threonine residues abolished amino acid editing activity. We propose that these threonine residues, which are also conserved in the homologous isoleucyl-tRNA synthetase and valyl-tRNA synthetase editing active sites, play a central role in amino acid editing. It is possible that they collaborate in stabilizing the transition state.

Acylation↗
PubMed2005

Structural probing of a pathogenic tRNA dimer.

The A3243G mutation within the human mitochondrial (hs mt) tRNALeuUUR gene is associated with maternally inherited deafness and diabetes (MIDD) and other mitochondrial encephalopathies. One of the most pronounced structural effects of this mutation is the disruption of the native structure through stabilization of a high-affinity dimeric complex. We conducted a series of studies that address the structural properties of this tRNA dimer, and we assessed its formation under physiological conditions. Enzymatic probing was used to directly define the dimeric interface for the complex, and a discrete region of the D-stem and loop of hs mt tRNALeuUUR was identified. The dependence of dimerization on magnesium ions and temperature was also tested. The formation of the tRNA dimer is influenced by temperature, with dimerization becoming more efficient at physiological temperature. Complexation of the mutant tRNA is also affected by the amount of magnesium present, and occurs at concentrations present intracellularly. Terbium probing experiments revealed a specific metal ion-binding site localized at the site of the A3243G mutation that is unique to the dimer structure. This metal ion-binding site presents a striking parallel to dimeric complexes of viral RNAs, which use the same hexanucleotide sequence for complexation and feature a similarly positioned metal ion-binding site within the dimeric structure. Taken together, these results indicate that the unique dimeric complex formed by the hs mt tRNALeuUUR A3243G mutant exhibits interesting similarities to biological RNA dimers, and may play a role in the loss of function caused by this mutation in vivo.

Base Sequence↗
PubMed2006

Acquisition of an insertion peptide for efficient aminoacylation by a halophile tRNA synthetase.

Enzymes of halophilic organisms contain unusual peptide motifs that are absent from their mesophilic counterparts. The functions of these halophile-specific peptides are largely unknown. Here we have identified an unusual peptide that is unique to several halophile archaeal cysteinyl-tRNA synthetases (CysRS), which catalyze attachment of cysteine to tRNA(Cys) to generate the essential cysteinyl-tRNA(Cys) required for protein synthesis. This peptide is located near the active site in the catalytic domain and is highly enriched with acidic residues. In the CysRS of the extreme halophile Halobacterium species NRC-1, deletion of the peptide reduces the catalytic efficiency of aminoacylation by a factor of 100 that largely results from a defect in kcat, rather than the Km for tRNA(Cys). In contrast, maintaining the peptide length but substituting acidic residues in the peptide with neutral or basic residues has no major deleterious effect, suggesting that the acidity of the peptide is not important for the kcat of tRNA aminoacylation. Analysis of general protein structure under physiological high salt concentrations, by circular dichroism and by fluorescence titration of tRNA binding, indicates little change due to deletion of the peptide. However, the presence of the peptide confers tolerance to lower salt levels, and fluorescence analysis in 30% sucrose reveals instability of the enzyme without the peptide. We suggest that the stability associated with the peptide can be used to promote proper enzyme conformation transitions in various stages of tRNA aminoacylation that are associated with catalysis. The acquisition of the peptide by the halophilic CysRS suggests an enzyme adaptation to high salinity.

Amino Acid Motifs↗
PubMed1978

Phosphonate analogues of aminoacyl adenylates.

Phosphonomethyl analogues of glycyl phosphate and valyl phosphate, i.e. NH2-CHR-CO-CH2-PO(OH)2, were synthesized and esterified with adenosine to give analogues of aminoacyl adenylates. The interaction of these adenylate analogues with valyl-tRNA synthetase from Escherichia coli was studied by fluorescence titration. The analogue of valyl phosphate has an affinity for the enzyme comparable with that of valine, but that of valyl adenylate is bound much less tightly than either valyl adenylate or corresponding derivative of valinol. The affinity of the analogue of glycyl adenylate was too low to be measured. We conclude that this enzyme interacts specifically with both the side chain and the anhydride linkage of the adenylate intermediate.

Adenosine Monophosphate↗
PubMed2003

[An insertion mutant of LeuRS with 116 amino acid residues has full activity].

Escherichia coli leucyl-tRNA synthetase (LeuRS) belongs to class I aminoacyl-tRNA synthetases. It consists of 860 amino acid residues and catalyzes the leucylation of tRNA(Leu). An insertion of its 253-368 peptide fragment between 368 to 369 in CP1 domain of this enzyme was shown to maintain the activity of the enzyme, and the insertion mutant was named as LeuRS-C. Because the insertion mutant of LeuRS was sensitive to operation of the purification, a plasmid containing the gene encoding LeuRS with His(6)-tag at its N-terminus was constructed to facilitate the purification of His(6)-LeuRS-C through one-step affinity chromatography on Ni(2+)-NTA column. The purified His(6)-LeuRS-C had full activity as the native LeuRS with His-tag at the N-terminus (His(6)-LeuRS), although the mutant enzyme had an insertion of 116 amino acid residues. The kinetic parameters of His(6)-LeuRS-C were determined. The secondary structure estimated by CD spectrum and thermal stability of the insertion mutant was compared with those of His(6)-LeuRS, respectively.

Circular Dichroism↗
PubMed1991

Functional analysis of the tobacco mosaic virus tRNA-like structure in cytoplasmic gene regulation.

The 3'-untranslated region (UTR) of tobacco mosaic virus (TMV), which terminates in a tRNA-like structure, functionally substitutes for a poly(A) tail in both plant and animal cells. The addition of the TMV 3'-UTR to chimeric mRNA constructs increases their expression up to 100-fold, increasing both translational efficiency and mRNA stability. The domain largely responsible for the regulation maps to a 72 base region immediately upstream of the tRNA-like structure, however, the 3'-terminal, tRNA-like structure is required for full function. Its contribution is lost if separated from the upstream pseudoknot domain by as few as 5 bases or if 6 bases are removed from the 3'-terminus. Sequence addition to the 3'-terminus of the TMV 3'UTR or the upstream pseudoknot domain inhibits function in both tobacco and Chinese hamster ovary cells.

Animals↗
PubMed1996

Efficient modification of RNA by porphyrin cation photochemistry: monitoring the folding of coaxially stacked RNA helices in tRNA(Phe) and the human immunodeficiency virus type 1 rev response element RNA.

Coaxially stacked RNA helices are a determined of RNA tertiary structure, but their presence is rarely detected using conventional chemical modification methods. In this report we describe a porphyrin ion photoreaction that enables one to monitor RNA stacking interactions and the folding of coaxially stacked RNA helices. The porphyrin cations meso-tetrakis(4-N-methylpyridyl)porphine, meso-tetrakis-(para-N-trimethylanilinium)porphine, and meso-tetrakis(2-N-methylpyridyl)porphine were used to characterize tRNA(Phe) and the human immunodeficiency virus type-I Rev response element RNA. Nucleosides at the bases of contiguous RNA helices in each RNA are efficiently modified by the porphyrin cations following irradiation of porphyrin-RNA mixtures. These photomodifications are markedly reduced for RNA equilibrated in ionic buffers that lead to enhanced stabilization of coaxially stacked helices. The porphyrin cation photoreaction specifically modifies G18, G20, and G34 in the tRNA folding produced by Mg(II). These nucleobases are exposed to solvent in the native tRNA structure and thus available to stack with solvent-borne porphyrin molecules. The describe porphyrin cation photochemical method provides a novel approach to study the solvent accessibility of nucleobases in RNA structure and to monitor the folding of coaxially stacked helices in RNA.

Aniline Compounds↗
PubMed2004

Molecular dissection of a critical specificity determinant within the amino acid editing domain of leucyl-tRNA synthetase.

A highly conserved threonine residue marks the amino acid binding pocket within the editing active site of leucyl-tRNA synthetases (LeuRSs). It is essential to substrate specificity for the Escherichia coli enzyme in that it blocks the cognate leucine amino acid from binding in the hydrolytic editing active site. We combined mutagenesis and computational approaches to elucidate the molecular role of the critical side chain of this threonine residue. Removal of the terminal methyl group of the threonine side chain by replacement with serine yielded a mutant LeuRS that hydrolyzes Leu-tRNA(Leu). Substitution of valine for the conserved threonine conferred similar activities to the wild-type enzyme. However, an additional substitution within the editing active site suggested synergistic interactions with the conserved threonine site that significantly affected amino acid editing. On the basis of our combined biochemical and computational data, we propose that the threonine 252 side chain not only sterically hinders the cognate charged leucine from binding for hydrolysis but also plays a critical role in maintaining an active site geometry that is required for the fidelity of LeuRS.

Amino Acid Substitution↗
PubMed2006

Mechanism of peptide bond formation on the ribosome.

Peptide bond formation is the fundamental reaction of ribosomal protein synthesis. The ribosome's active site--the peptidyl transferase center--is composed of rRNA, and thus the ribosome is the largest known RNA catalyst. The ribosome accelerates peptide bond formation by 10(7)-fold relative to the uncatalyzed reaction. Recent progress of structural, biochemical and computational approaches has provided a fairly detailed picture of the catalytic mechanisms employed by the ribosome. Energetically, catalysis is entirely entropic, indicating an important role of solvent reorganization, substrate positioning, and/or orientation of the reacting groups within the active site. The ribosome provides a pre-organized network of electrostatic interactions that stabilize the transition state and facilitate proton shuttling involving ribose hydroxyl groups of tRNA. The catalytic mechanism employed by the ribosome suggests how ancient RNA-world enzymes may have functioned.

Binding Sites↗
PubMed1984

tRNA binding sites of ribosomes from Escherichia coli.

70S tight-couple ribosomes from Escherichia coli were studied with respect to activity and number of tRNA binding sites. The nitrocellulose filtration and puromycin assays were used both in a direct manner and in the form of a competition binding assay, the latter allowing an unambiguous determination of the fraction of ribosomes being active in tRNA binding. It was found that, in the presence of poly(U), the active ribosomes bound two molecules of N-AcPhe-tRNAPhe, one in the P and the other in the A site, at Mg2+ concentrations between 6 and 20 mM. A third binding site in addition to P and A sites was observed for deacylated tRNAPhe. At Mg2+ concentrations of 10 mM and below, the occupancy of the additional site was very low. Dissociation of tRNA from this site was found to be rather fast, as compared to both P and A sites. These results suggest that the additional site during translocation functions as an exit site, to which deacylated tRNA is transiently bound before leaving the ribosome. Since tRNA binding to this site did not require the presence of poly(U), a function of exit site bound tRNA in the fixation of the mRNA appears unlikely. Both the affinity and stability of binding to the additional site were found lower for the heterologous tRNAPhe from yeast as compared to the homologous one. This difference possibly indicates some specificity of the E. coli ribosome for tRNAs from the same organism.

Binding Sites↗
PubMed2003

The tau95 subunit of yeast TFIIIC influences upstream and downstream functions of TFIIIC.DNA complexes.

The yeast transcription factor IIIC (TFIIIC) is organized in two distinct multisubunit domains, tauA and tauB, that are respectively responsible for TFIIIB assembly and stable anchoring of TFIIIC on the B block of tRNA genes. Surprisingly, we found that the removal of tauA by mild proteolysis stabilizes the residual tauB.DNA complexes at high temperatures. Focusing on the well conserved tau95 subunit that belongs to the tauA domain, we found that the tau95-E447K mutation has long distance effects on the stability of TFIIIC.DNA complexes and start site selection. Mutant TFIIIC.DNA complexes presented a shift in their 5' border, generated slow-migrating TFIIIB.DNA complexes upon stripping TFIIIC by heparin or heat treatment, and allowed initiation at downstream sites. In addition, mutant TFIIIC.DNA complexes were highly unstable at high temperatures. Coimmunoprecipitation experiments indicated that tau95 participates in the interconnection of tauA with tauB via its contacts with tau138 and tau91 polypeptides. The results suggest that tau95 serves as a scaffold critical for tauA.DNA spatial configuration and tauB.DNA stability.

Amino Acid Sequence↗
PubMed1987

Formation of delta-Aminolevulinic Acid from Glutamic Acid in Algal Extracts : Separation into an RNA and Three Required Enzyme Components by Serial Affinity Chromatography.

Extracts from plant chloroplasts and algae catalyze the conversion of glutamate to delta-aminolevulinic acid (ALA) in the first committed step of the tetrapyrrole biosynthetic pathway leading to chlorophylls, hemes, and bilins. The conversion requires ATP, Mg(2+), and NADPH as cofactors. Soluble extracts from Chlorella vulgaris have now been resolved into four macromolecular fractions, all of which are required to reconstitute activity. One fraction contains a low molecular weight RNA which can be separated from the protein components in an active high-speed supernatant by treatment with 1 molar NaCl followed by precipitation of the proteins with (NH(4))(2)SO(4) at 70% saturation. The proteins recovered from the (NH(4))(2)SO(4) precipitate are reactivated by addition of a fraction containing tRNAs isolated from Chlorella by phenol-chloroform extraction and DEAE cellulose chromatography. Three required protein fractions were resolved from the RNA-depleted (NH(4))(2)SO(4) precipitate by serial affinity chromatography on Reactive Blue 2-Sepharose and 2',5'-ADP-agarose. Glycerol was found to stabilize the enzyme activity during the separation process. The majority of the glutamate:tRNA ligase activity was associated with the fraction which was retained by Blue-Sepharose and not retained by ADP-agarose, in agreement with the reported properties of the affinity ligands. The active material in the fraction not retained by Blue-Sepharose eluted as a single component on gel filtration chromatography, with an apparent molecular weight of 67,000. The active component in the RNA fraction also eluted as a single component on gel filtration chromatography.

Journal Article↗
PubMed1997

Characterization of Neurospora mitochondrial group I introns reveals different CYT-18 dependent and independent splicing strategies and an alternative 3' splice site for an intron ORF.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing the N. crassa mitochondrial large rRNA intron by stabilizing the catalytically active structure of the intron core. Here, a comprehensive study of N. crassa mtDNA group I introns identified two additional introns, cob-I2 and the ND1 intron, that are dependent on CYT-18 for splicing in vitro and in vivo. The other seven N. crassa mtDNA group I introns are not CYT-18-dependent and include five that self-splice and two that do not splice under any conditions examined. Some of these introns may require maturases or other proteins for efficient splicing. All but one of the non-CYT-18-dependent introns contain large peripheral extensions of the P5 stem, related to the P5abc structure that blocks CYT-18 binding to the Tetrahymena large rRNA intron. The remaining non-CYT-18-dependent intron, cob-I1, contains a long, peripheral extension of the P9 stem, denoted P9.1, which also impedes CYT-18 binding. Detailed analysis of the CYT-18-dependent ND1 intron showed that two 3' splice sites are used in vitro and in vivo. The proximal, alternative 3' splice site brings the intron open reading frame, which potentially encodes a mobility endonuclease, in frame with the upstream exon, possibly providing a means of expression. Considered together, our results show that group I introns in N. crassa mitochondria use a variety of strategies involving different proteins and/or RNA structures to assist splicing, and they support the hypothesis that CYT-18 and the peripheral RNA structure P5abc are alternative evolutionary adaptations for stabilizing the active structure of the intron core.

Alternative Splicing↗
PubMed1995

Increased rates of tRNA charging through modification of the enzyme-aminoacyl-adenylate complex of phenylalanyl-tRNA synthetase.

The transfer of amino acid to tRNA by Escherichia coli phenylalanyl-tRNA synthetase (PheRS) was studied using replacements of Ala294 in the alpha subunit previously shown to have modified amino acid specificity. Steady-state analysis of tRNA charging showed little difference between wild-type and mutants, whereas pre-steady-state analysis revealed higher rates of tRNA charging by both the A294S PheRS-phenylalanyl adenylate and the A294G PheRS-p-Cl-phenylalanyl adenylate. The decrease in energy required for the formation of the transition state of amino acid transfer in these mutants could be related to a weaker binding of the amino acid in the aminoacyl adenylate complex. Thus a compromise appears to exist between amino acid activation and tRNA charging, because slowing down the first step increases the rate of the second step, possibly as a result of decreased stability of the PheRS.amino acid-AMP complex.

Adenosine Monophosphate↗