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At least 703 records · Page 39Linked to original sources
PubMed2004

Mutation and evolution of the magnesium-binding site of a class II aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetases contain one or three Mg(2+) ions in their catalytic sites. In addition to their role in ATP binding, these ions are presumed to play a role in catalysis by increasing the electropositivity of the alpha-phosphate and stabilizing the pentavalent transition state. In the class II aaRS, two highly conserved carboxylate residues have been shown to participate with Mg(2+) ions in binding and coordination. It is shown here that these carboxylate residues are absolutely required for the activity of Saccharomyces cerevisiae aspartyl-tRNA synthetase. Mutants of these residues exhibit pleiotropic effects on the kinetic parameters suggesting an effect at an early stage of the aminoacylation reaction, such as the binding of ATP, Mg(2+), aspartic acid, or the amino acid activation. Despite genetic selections in an APS-knockout yeast strain, we were unable to select a single active mutant of these carboxylate residues. Nevertheless, we isolated an intragenic suppressor from a combinatorial library. The active mutant showed a second substitution close to the first one, and exhibited a significant increase of the tRNA aminoacylation rate. Structural analysis suggests that the acceptor stem of the tRNA might be repositioned to give a more productive enzyme:tRNA complex. Thus, the initial defect of the activation reaction was compensated by a significant increase of the aminoacylation rate that led to cellular complementation.

Acylation↗
PubMed2005

Structural basis for autoinhibition and mutational activation of eukaryotic initiation factor 2alpha protein kinase GCN2.

The GCN2 protein kinase coordinates protein synthesis with levels of amino acid stores by phosphorylating eukaryotic translation initiation factor 2. The autoinhibited form of GCN2 is activated in cells starved of amino acids by binding of uncharged tRNA to a histidyl-tRNA synthetase-like domain. Replacement of Arg-794 with Gly in the PK domain (R794G) activates GCN2 independently of tRNA binding. Crystal structures of the GCN2 protein kinase domain have been determined for wild-type and R794G mutant forms in the apo state and bound to ATP/AMPPNP. These structures reveal that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. The R794G mutant shows increased flexibility in the hinge region connecting the N- and C-lobes, resulting from loss of multiple interactions involving Arg794. This conformational change is associated with intradomain movement that enhances ATP binding and hydrolysis. We propose that intramolecular interactions following tRNA binding remodel the hinge region in a manner similar to the mechanism of enzyme activation elicited by the R794G mutation.

Adenosine Triphosphate↗
PubMed1988

Domain VI of Escherichia coli 23 S ribosomal RNA. Structure, assembly and function.

Domain VI at the 3' end of the 23 S ribosomal RNA from Escherichia coli was prepared using the in vitro T7 RNA polymerase system. Its structure was examined by probing with ribonucleases and chemical reagents, including a psoralen derivative, of various nucleotide specificities, using a reverse transcriptase procedure for analysis. The data provided support for the most recent secondary structure derived from phylogenetic sequence comparisons and for additional structuring that was inferred from earlier experimental data. Moreover, the structure was essentially the same in the free domain, in renatured 23 S RNA and in 50 S subunits. Protein L3 bound to the isolated domain and its binding site was located at a long-range double helix containing a large internal loop. This structure is unusual for a protein-RNA binding site and it may characterize a new (third) class of site. Protein L3 has been implicated, together with L24, in initiating assembly of the 50 S subunit and it shares the exceptional property with L24 that it binds adjacent to the junction of two RNA domains from where it can maximally influence RNA folding. Protein L6 also assembled to domain VI and, in a control experiment, protein L2 bound to isolated domain IV. Domain VI was largely inaccessible in the 50 S subunit and the few accessible RNA sites occurred mainly within conserved sequence regions that constitute potential functional sites. alpha-Sarcin inactivates ribosomes by cutting at one of these sites in 50 S subunits; it also recognized the same site in the free 23 S RNA and in the free domain. Both the EF-Tu ternary complex, and the EF-G ternary complex stabilized by fusidic acid or by a non-hydrolyzable GTP derivative, inhibited alpha-sarcin attack while non-enzymatically bound tRNA did not, thus providing evidence, more direct than before, for the involvement of the RNA region in a common elongation factor binding site.

Autoradiography↗
PubMed1988

Model for the three-dimensional folding of 16 S ribosomal RNA.

We have derived a model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA, using interactive computer graphic methods. It is based on (1) the secondary structure derived from comparative sequence analysis, (2) the three-dimensional co-ordinates for the centers of mass of the 30 S subunit proteins, and (3) the locations of sites in 16 S rRNA that interact with specific ribosomal proteins, from footprinting and crosslinking studies. We present a detailed description of the derivation of the model. About 75% of the RNA chain is sufficiently constrained to provide a useful model. This contains most of the universally conserved core of the molecule. In all but a few instances, protected and crosslinked sites can be placed within or very close to their cognate proteins, while obeying stereochemical rules. The overall shape of the model and locations of specific regions of the RNA correspond well to data derived from electron micrographs of 30 S subunits, although such data were not used to construct the model. Phylogenetic variations in the structure are readily accommodated; as an example, we have modeled the 950-nucleotide mammalian mitochondrial 12 S rRNA by superimposing it on the E. coli structure. The three major RNA domains, as defined by secondary structure, appear to exist as autonomous structural units in three dimensions, for the most part. There is an extensive interface between the 5' and central domains, whereas the 3' major domain has relatively little apparent contact with the rest of the structure. The 5', central and 3' major domains form structures that resemble the body, platform and head, respectively, seen in electron micrographs of 30 S subunits. We discuss possible roles for the ribosomal proteins in stabilizing specific structural features of the RNA during ribosome assembly. The decoding site, as deduced from footprinting and crosslinking studies involving the tRNA anticodon stem-loop, is well-localized. Bases protected from chemical probing by the anticodon stem-loop line the cleft of the subunit. The conserved loop at position 530, which contains some of the bases protected by A site-bound tRNA, is remote (approx. 80 A) from the decoding site. Protection of these bases by the anticodon stem-loop is thus unlikely to be due to direct contact.

Base Sequence↗
PubMed1992

Design of peptide-acridine mimics of ribonuclease activity.

A series of peptide-acridine conjugates was designed and synthesized, based on three features of the proposed catalytic mechanism of RNase A: 2'-proton abstraction by His-12, proton donation to the leaving 5'-oxygen by His-119, and stabilization of the pentacoordinated phosphorous transition state by Lys-41. The substrate binding capability of RNase A was mimicked by the intercalator, acridine. Lysine served as a linker between acridine and the catalytic tripeptide. Cleavage of target RNA was monitored by agarose gel electrophoresis and by gel-permeation chromatography. The carboxyl-amidated conjugates HGHK(Acr)-NH2, HPHK(Acr)-NH2, and GGHK(Acr)-NH2 (where Acr indicates 2-methyl-9-acridinemethylene) all had similar hydrolytic activity. The catalytic mechanism most likely involved only the abstraction of the 2'-proton and stabilization of the transition state. These RNase mimics utilized rRNA and single-stranded RNA but not double-stranded RNA and tRNA as substrates.

Acridines↗
PubMed2000

Active aminoacyl-tRNA synthetases are present in nuclei as a high molecular weight multienzyme complex.

Recent studies suggest that aminoacylation of tRNA may play an important role in the transport of these molecules from the nucleus to the cytoplasm. However, there is almost no information regarding the status of active aminoacyl-tRNA synthetases within the nuclei of eukaryotic cells. Here we show that at least 13 active aminoacyl-tRNA synthetases are present in purified nuclei of both Chinese hamster ovary and rabbit kidney cells, although their steady-state levels represent only a small percentage of those found in the cytoplasm. Most interestingly, all the nuclear aminoacyl-tRNA synthetases examined can be isolated as part of a multienzyme complex that is more stable, and consequently larger, than the comparable complex isolated from the cytoplasm. These data directly demonstrate the presence of active aminoacyl-tRNA synthetases in mammalian cell nuclei. Moreover, their unexpected structural organization raises important questions about the functional significance of these multienzyme complexes and whether they might play a more direct role in nuclear to cytoplasmic transport of tRNAs.

Active Transport, Cell Nucleus↗
PubMed2000

Prediction of common secondary structures of RNAs: a genetic algorithm approach.

In this study we apply a genetic algorithm to a set of RNA sequences to find common RNA secondary structures. Our method is a three-step procedure. At the first stage of the procedure for each sequence, a genetic algorithm is used to optimize the structures in a population to a certain degree of stability. In this step, the free energy of a structure is the fitness criterion for the algorithm. Next, for each structure, we define a measure of structural conservation with respect to those in other sequences. We use this measure in a genetic algorithm to improve the structural similarity among sequences for the structures in the population of a sequence. Finally, we select those structures satisfying certain conditions of structural stability and similarity as predicted common structures for a set of RNA sequences. We have obtained satisfactory results from a set of tRNA, 5S rRNA, rev response elements (RRE) of HIV-1 and RRE of HIV-2/SIV, respectively.

Algorithms↗
PubMed1996

Strategies for achieving high-level expression of genes in Escherichia coli.

Progress in our understanding of several biological processes promises to broaden the usefulness of Escherichia coli as a tool for gene expression. There is an expanding choice of tightly regulated prokaryotic promoters suitable for achieving high-level gene expression. New host strains facilitate the formation of disulfide bonds in the reducing environment of the cytoplasm and offer higher protein yields by minimizing proteolytic degradation. Insights into the process of protein translocation across the bacterial membranes may eventually make it possible to achieve robust secretion of specific proteins into the culture medium. Studies involving molecular chaperones have shown that in specific cases, chaperones can be very effective for improved protein folding, solubility, and membrane transport. Negative results derived from such studies are also instructive in formulating different strategies. The remarkable increase in the availability of fusion partners offers a wide range of tools for improved protein folding, solubility, protection from proteases, yield, and secretion into the culture medium, as well as for detection and purification of recombinant proteins. Codon usage is known to present a potential impediment to high-level gene expression in E. coli. Although we still do not understand all the rules governing this phenomenon, it is apparent that "rare" codons, depending on their frequency and context, can have an adverse effect on protein levels. Usually, this problem can be alleviated by modification of the relevant codons or by coexpression of the cognate tRNA genes. Finally, the elucidation of specific determinants of protein degradation, a plethora of protease-deficient host strains, and methods to stabilize proteins afford new strategies to minimize proteolytic susceptibility of recombinant proteins in E. coli.

Biotechnology↗
PubMed1999

Mitochondrial DNA acts as potential promoter of the baculovirus RNA polymerase.

We have examined whether mitochondrial DNA could act as target of the RNA polymerase encoded by the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus, because the baculovirus late promoters and the control region of host mitochondrial DNA show a high degree of sequence similarity. In vitro transcription using mitochondrial DNA from Spodoptera frugiperda cells and nuclear extracts prepared from baculovirus infected cells demonstrates that mitochondrial DNA is recognized by the viral RNA polymerase. Transcriptional initiation occurs at TAAG sequences, although not all of the six TAAG motifs present in the mitochondrial DNA fragment are recognized. The TAAG motif in the control region served as weak transcriptional start site, but some of the TAAG motifs in the coding sequences of the adjacent tRNA and rRNA genes are recognized efficiently. The sequences flanking the TAAG motifs used as transcriptional start sites have a lower helix stability than the flanking sequences of the nonfunctional TAAG motifs. These results support the view that helix stability rather than sequence specificity is an important factor for recognition of TAAG motifs by the viral RNA polymerase.

Animals↗
PubMed1995

GNRA tetraloops make a U-turn.

The U-turn (uridine turn) is an RNA structural motif that contains a change in backbone direction stabilized by specific interactions across the bend. It was first identified in the anticodon loop and the T-loop of yeast tRNA(Phe) (Quigley & Rich, 1976, Science 194:796-806) and has recently also been found in the crystal structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB, 1994a, Nature 372:68-74). These U-turn motifs follow a UNR consensus sequence (where N is any nucleotide and R is G or A). Here we report that the frequently occurring GNRA tetraloops also contain a U-turn motif, and we discuss the role of U-turns as abundant tertiary structural motifs in RNA.

Nucleic Acid Conformation↗
PubMed2004

Metal ion stabilization of the U-turn of the A37 N6-dimethylallyl-modified anticodon stem-loop of Escherichia coli tRNAPhe.

Nucleoside base modifications can alter the structures, dynamics, and metal ion binding properties of transfer RNA molecules and are important for accurate aminoacylation and for maintaining translational fidelity and efficiency. The unmodified anticodon stem-loop from Escherichia coli tRNA(Phe) forms a trinucleotide loop in solution, but Mg(2+) and dimethylallyl modification of A(37) N6 disrupt the loop conformation and increase the mobility of the loop and loop-proximal nucleotides. We have used NMR spectroscopy to investigate the binding and structural effects of multivalent cations on the unmodified and dimethylallyl-modified anticodon stem-loops from E. coli tRNA(Phe). The divalent cation binding sites were probed using Mn(2+) and Co(NH(3))(6)(3+). These ions bind along the major groove of the stem and associate with the anticodon loop on the major groove side in a nonspecific manner. Co(NH(3))(6)(3+) stabilizes the U-turn conformation of the loop in the dimethylallyl-modified molecule, and the chemical shift changes that accompany Co(NH(3))(6)(3+) binding are similar to those observed with the addition of Mg(2+). The base-phosphate and base-2'-OH hydrogen bonds that characterize the UNR U-turn motif lead to spectral signatures in the form of unusual (15)N and (1)H chemical shifts and reduced solvent exchange of the U(33) 2'-OH and N3H protons. The unmodified molecule also displays spectral features of the U-turn fold in the presence of Co(NH(3))(6)(3+), but the loop has additional conformations and is dynamic. The results indicate that charge neutralization by a polyvalent cation is sufficient to promote formation of the U-turn fold. However, base modification is necessary to destabilize competing alternative conformers even for a purine-rich loop sequence that is predicted to have strongly favorable base stacking energy.

Adenine↗
PubMed1992

Novel transfer RNAs that are active in Escherichia coli.

Many of the mammalian mitochondrial tRNAs contain significant nucleotide deletions in the dihydrouridine (D) stem or T psi C stem, so that they cannot fold into the canonical cloverleaf structure. This suggests that alternative forms and shapes are possible for a mitochondrial tRNA that functions in the specialized translational apparatus of the mammalian mitochondria. The question of whether significant structural alterations may be accommodated by a bacterial protein synthesis machinery, such as in Escherichia coli, is unanswered. In this work, all but ten positions in the gene for the 76-nucleotide coding sequence of an E. coli amber suppressor tRNA were permuted and screened for biological activity in vivo. Sequence analysis of a collection of biologically active variants established that many have unusual structures that include base-pair mismatches in helical stems, substitutions of normally conserved bases, and deletions. Independent mutations were obtained that weaken base pairs or tertiary interactions that normally stabilize the coaxial stacking of the D and anticodon stems, suggesting that the translational apparatus can accommodate considerable flexibility in this part of the molecule. The results demonstrate the capacity of the bacterial protein synthetic apparatus to accommodate altered tRNA structures that are not represented by any naturally occurring tRNAs.

Base Sequence↗
PubMed1977

Ternary complex formation between elongation factor Tu, GTP and aminoacyl-tRNA: an equilibrium study.

The equilibria between the elongation factor Tu-GTP complex (EF-Tu-GTP) from Escherichia coli and tyrosyl-tRNATyr from E. coli as well as phenylalanyl-tRNAPhe and seryl-tRNASer from yeast were studied using a novel procedure, which takes advantage of the protective effect of ternary complex formation on the stability of theaminoacyl bond against non-enzymatic hydrolysis. At 25 degrees C and at pH 7.4 tyrosyl-tRNATyr, phenylalanyl-tRNAPhe and seryl-tRNASer are bound with binding constants of 0.7 X 10(7) M-1, 5.0 X 10(7) M-1 and 0.5 X 10(7) M-1 respectively. The binding of aminoacyl-tRNA to EF-Tu-GTP has a negative deltaH of the order of 10 kcal/mol (42 kJ/mol). Complex formation is dependent on ionic strength: with 0.1 M KCl Kass = 0.8 X 10(7) M-1, with 0.5 M KCl Kass = 0.2 X 10(7) M-1 was determined for the binding of Tyr-tRNATyr.

Calorimetry↗
PubMed1981

Synthesis of a nitrobenzeneboronic acid substituted polyacrylamide and its use in purifying isoaccepting transfer ribonucleic acids.

Highly purified isoaccepting species of transfer ribonucleic acid (tRNA) were prepared by use of a polyacrylamide substituted with nitrobenzeneboronic acid functional groups. This method exploits the well-known ability of boronic acids to complex with RNA cis-diols. tRNA isoacceptors were obtained by enzymatically acylating a mixture of tRNA species with a single amino acid and passing the mixture over a solid-state nitrobenzeneboronic acid at pH 6.5 or 7.0. Pure aminoacyl-tRNA eluted at the column liquid volume, and unacylated tRNA species were bound. The bound species were recovered by lowering the pH of the eluant to 4.5. This procedure is uncomplicated, rapid, and applicable to nearly all tRNA isoacceptors. It does not chemically modify the tRNA(s) of interest or adversely affect their ability to be aminoacylated. Since boronic acids must be ionized to complex with cis-1,2-diols, boronic acid derivatives were prepared which ionize at a pH compatible with the stability of the aminoacyl bond. Two isomeric benzeneboronic acids with pKas of 6.8 and 7.4 were synthesized by introducing electron-withdrawing nitro groups into the aromatic ring. The addition of succinyl side chains permitted the nitrobenzeneboronic acids to be coupled to aminoethylpolyacrylamide. The properties of the nitrobenzeneboronic acid substituted acrylamide were illustrated by enriching phenylalanyl-tRNA at pH 7.0 to greater than 95% purity (1.63 nmol of phenylalanine accepted per A260 unit of tRNA) and seryl-tRNA isoacceptors at pH 6.5 to essentially theoretical purity (1.58 nmol of serine accepted per A260 unit of tRNA.

Acrylic Resins↗