Knocking out a specific tRNA species within unfractionated Escherichia coli tRNA by using antisense (complementary) oligodeoxyribonucleotides

Methods for the preparation of an Escherichia coli tRNA mixture lacking one or a few specific tRNA species can be the basis for future applications of cell-free protein synthesis. We demonstrate here that virtually a single tRNA species in a crude E. coli tRNA mixture can be knocked out by an antisense (complementary) oligodeoxyribonucleotide. One out of five oligomers complementary to tRNA^Asp blocked the aspartylation almost completely, while minimally affecting the aminoacylation with other 13 amino acids tested. This `knockout' tRNA behaved similarly to the untreated tRNA in a cell-free translation of an mRNA lacking Asp codons.     

Genetic analysis of functional connectivity between substrate recognition domains ofEscherichia coli glutaminyl-tRNA synthetase

It has previously been shown that the single mutation E222K in glutaminyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype onEscherichia coli. Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to investigate the role of these residues in substrate discrimination. The three-dimensional structure of the tRNA^Gln: GlnRS:ATP ternary complex revealed that both E222 and C171 are close to regions of the protein involved in interactions with both the acceptor stem and the 3 end of tRNA^Gln. The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischargesupF tRNA^Tyr considerably more efficiently than the wild-type enzyme, whereas GlnRS-E222K/C171G could not. These differences in substrate specificity also extended to anticodon recognition, with the double mutant able to distinguishsupE tRNA _CUA ^Gln from tRNA ₂ ^Gln considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E222K was found to have a 15-fold higher K_m for glutamine than the wild-type enzyme, whereas the double mutant only showed a 7-fold increase. These results indicate that the C171G mutation improves both substrate discrimination and recognition at three domains in GlnRS-E222K, confirming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.     

Protein Modification by RNA-Dependent Posttranslational Aminoacylation in Synaptoplasm

A soluble enzyme system that posttranslationally adds [3H]arginine to proteins in a ribosome-free preparation of guinea pig synaptoplasm is described. The reaction in synaptoplasm is inhibited by the addition of ribonuclease-A and puromycin, indicating tRNA dependence. A limited number of proteins in synaptoplasm (molecular weights of 20, 37, and 50 kilodaltons) were found to accept arginine. We suggest that RNA-dependent posttranslational amino acylation is used by the mammalian neuron for protein processing at the synaptic terminal.     

The balance between pre- and post-transfer editing in tRNA synthetases

The fidelity of tRNA aminoacylation is dependent in part on amino acid editing mechanisms. A hydrolytic activity that clears mischarged tRNAs typically resides in an active site on the tRNA synthetase that is distinct from its synthetic aminoacylation active site. A second pre-transfer editing pathway that hydrolyzes the tRNA synthetase aminoacyl adenylate intermediate can also be activated. Pre- and post-transfer editing activities can co-exist within a single tRNA synthetase resulting in a redundancy of fidelity mechanisms. However, in most cases one pathway appears to dominate, but when compromised, the secondary pathway can be activated to suppress tRNA synthetase infidelities.     

Mild, rapid and selective alcoholysis of terpyridine-appended amide substrates by Cu-catalysis: protonation state and reactivity of the complex

An amide bond of a terpyridine-appended substrate, 6-(L-phenylalanylamino)-2,2′:6′,2″-terpyridine (2a), was cleaved to yield phenylalanine methyl ester quantitatively in the presence of catalytic amounts of Cu²⁺ not only in methanol but also in aqueous methanol at 30 °C. The reaction proceeds via formation of an N₃O (three terpyridine nitrogens and one carbonyl oxygen) type 1:1 metal complex 2a-Cu²⁺. From spectral titration, the structure of the 2a-Cu²⁺ complex was confirmed to have three different protonation states, i.e., A (non-deprotonated amide with α-ammonium), D1 (deprotonated-amide with α-ammonium) and D2 (deprotonated-amide with α-amino) states. Among them, the complex in the D2 state was exclusively responsible for the observed mild, rapid and selective alcoholysis, showing the first-order rate constant of 6 × 10⁻³ s⁻¹ or half-decay time of 2 min in methanol at 30 °C. Acidity of the amide proton was found to be higher than that of the ammonium proton in the complex, allowing formation of the highly reactive amide-deprotonated D2 state in methanol even without addition of external bases. Factors contributing to the high reactivity of the complex were discussed.     

Inter-chain Proline: Proline Contacts Contribute to the Stability of the Triple Helical Conformation

Abstract

The triple helical conformation observed in the collagen group of proteins is related to the presence of large numbers of imino residues and is derived from the stereochemical properties of these residues. The triple helix is stabilized by increasing numbers of these residues. Hydrogen bonds are usually considered to be a major factor in the formation and stability of protein conformation, however, imino residues are not hydrogen bond donors. We have evaluated the role of these residues in stabilizing the triple helix by re-examining two X-ray based structures of the triple helical polypeptide (Pro-Pro- Gly)₁₀ using molecular mechanics calculations. The two minimized structures are comparable in energy and have helical parameters close to the starting values for each starting structure. Our studies suggest that clusters of close van der Waals contacts between proline residues in adjacent chains contribute significantly to the stability of the triple helix. Preliminary NMR studies support this concept. We propose that non-bonded interactions between proline residues may be a significant stabilizing force in the triple helix generated by (Pro-Pro-Gly)₁₀.     

Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog

Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs.     

Phosphoserine aminoacylation of tRNA bearing an unnatural base anticodon

An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a K_m = 47.1 μM and a k_cat = 0.151 s⁻¹, which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNA^Cys with the GCA anticodon (26.9 μM and 0.111 s⁻¹). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.     

Crystallization and preliminary X-ray diffraction analysis of <Emphasis Type="Italic">E. coli</Emphasis> arginyl-tRNA synthetase in complex form with a tRNA<Superscript>Arg</Superscript>

Amino acids are building blocks of proteins, while aminoacyl-tRNA synthetases (aaRSs) catalyze the first reaction in such building: the biosynthesis of proteins. The E. coli arginyl-tRNA synthetase (ArgRS) has been crystallized in complex form with tRNA(Arg) (B. stearothermophilus), at pH 5.6 using ammonium sulfate as a precipitating agent. Two crystal forms have been identified based on unit cell dimension. The complete data sets from both crystal forms have been collected with a primitive hexagonal space group. A data set of Form II crystals at 3.2 A and 94% completeness has been obtained, with unit cell parameters a = b = 98.0 A, c = 463.2 A, and alpha = beta = 90 degrees , gamma = 120 degrees , being different from a = b = 110.8 A, c = 377.8 A for form I. The structure determination will demonstrate the interaction of these two macromolecules to understand the special mechanism of ArgRS that requires the presence of tRNA for amino acid activation. Such complex structure also provides a wide opening for inhibitor search using bioinformatics.