Searching tRNA sequences for relatedness to aminoacyl-tRNA synthetase families

tRNA sequences were analyzed for sequence features correlated with known classes of aminoacyl-tRNA synthetase enzymes. The tRNAs were searched for distinguishing nucleotides anywhere in their sequences. The analyses did not find nucleotides predictive of synthetase class membership. We conclude that such nucleotides never existed in tRNA sequences or that they existed and were lost from many of the tRNA sequences during evolution.Based on a presentation made at a workshop—Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code—held at Berkeley, CA, July 17–20, 1994 Correspondence to: H.B. Nicholas, Jr.     

tRNA discrimination at the binding step by a class II aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accurately joining amino acids to their cognate transfer RNAs. Here, tRNA discrimination at the level of binding by Escherichia coli histidyl-tRNA synthetase is addressed by filter binding, analytical ultracentrifugation, and iodine footprinting experiments. Competitive filter binding assays show that the presence of an adenylate analogue 5'-O-[N-(L-histidyl)sulfamoyl]adenosine, HSA, decreased the apparent dissociation constant (K(D)) for cognate tRNA(His) by more than 3-fold (from 3.87 to 1.17 microM), and doubled the apparent K(D) for noncognate tRNA(Phe) (from 7.3 to 14.5 microM). By contrast, no binding discrimination against mutant U73 tRNA(His) was observed, even in the presence of HSA. Additional filter binding studies showed tighter binding of both cognate and noncognate tRNAs by G405D mutant HisRS [Yan, W., Augustine, J., and Francklyn, C. (1996) Biochemistry 35, 6559], which possesses a single amino acid change in the C-terminal anticodon binding domain. Discrimination against noncognate tRNA was also observed in sedimentation velocity experiments, which showed that a stable complex was formed with the cognate tRNA(His) but not with noncognate tRNA(Phe). Footprinting experiments on wild-type versus G405D HisRS revealed characteristic alterations in the pattern of protection and enhancement of iodine cleavage at phosphates 5' to tRNA nucleotides in the anticodon and hinge regions. Together, these results suggest that the anticodon and core regions play major roles in the initial binding discrimination between cognate and noncognate tRNAs, whereas acceptor stem nucleotides, particularly at position 73, influence the reaction at steps after binding of tRNA.     

Kinetic discrimination of tRNA identity by the conserved motif 2 loop of a class II aminoacyl-tRNA synthetase

The selection of tRNAs by their cognate aminoacyl-tRNA synthetases is critical for ensuring the fidelity of protein synthesis. While nucleotides that comprise tRNA identity sets have been readily identified, their specific role in the elementary steps of aminoacylation is poorly understood. By use of a rapid kinetics analysis employing mutants in tRNA(His) and its cognate aminoacyl-tRNA synthetase, the role of tRNA identity in aminoacylation was investigated. While mutations in the tRNA anticodon preferentially affected the thermodynamics of initial complex formation, mutations in the acceptor stem or the conserved motif 2 loop of the tRNA synthetase imposed a specific kinetic block on aminoacyl transfer and decreased tRNA-mediated kinetic control of amino acid activation. The mechanistic basis of tRNA identity is analogous to fidelity control by DNA polymerases and the ribosome, whose reactions also demand high accuracy.     

Limited set of amino acid residues in a class Ia aminoacyl-tRNA synthetase is crucial for tRNA binding

The aim of this work was to characterize crucial amino acids for the aminoacylation of tRNA(Arg) by yeast arginyl-tRNA synthetase. Alanine mutagenesis was used to probe all the side chain mediated interactions that occur between tRNA(Arg2)(ICG) and ArgRS. The effects of the substitutions were analyzed in vivo in an ArgRS-knockout strain and in vitro by measuring the aminoacylation efficiencies for two distinct tRNA(Arg) isoacceptors. Nine mutants that generate lethal phenotypes were identified, suggesting that only a limited set of side chain mediated interactions is essential for tRNA recognition. The majority of the lethal mutants was mapped to the anticodon binding domain of ArgRS, a helix bundle that is characteristic for class Ia synthetases. The alanine mutations induce drastic decreases in the tRNA charging rates, which is correlated with a loss in affinity in the catalytic site for ATP. One of those lethal mutations corresponds to an Arg residue that is strictly conserved in all class Ia synthetases. In the known crystallographic structures of complexes of tRNAs and class Ia synthetases, this invariant Arg residue stabilizes the idiosyncratic conformation of the anticodon loop. This paper also highlights the crucial role of the tRNA and enzyme plasticity upon binding. Divalent ions are also shown to contribute to the induced fit process as they may stabilize the local tRNA-enzyme interface. Furthermore, one lethal phenotype can be reverted in the presence of high Mg(2+) concentrations. In contrast with the bacterial system, in yeast arginyl-tRNA synthetase, no lethal mutation has been found in the ArgRS specific domain recognizing the Dhu-loop of the tRNA(Arg). Mutations in this domain have no effects on tRNA(Arg) aminoacylation, thus confirming that Saccharomyces cerevisiae and other fungi belong to a distinct class of ArgRS.     

Identification of an apparent aminoacyl-tRNA synthetase activator factor as tRNA nucleotidyltransferase

Summary The ability of yeast extracts to aminoacylate crude yeast tRNA with leucine and other amino acids is largely lost after chromatography of the extracts in DEAE-Sephadex. The original aminoacylating ability is restored by combining protein fractions from the DEAE-chromatogram. The characteristics of this reactivation are very similar to the activation, by protein factors, of certain aminoacyl-tRNA synthetases reported by others. The results in this work indicate that the apparent aminoacyl-tRNA synthetase activator factor is the tRNA nucleotidyltransferase and that the restoration of the original tRNA aminoacylating ability is a consequence of the repairing of the 3' end of incomplete tRNA chains.     

Electrostatic potential of aminoacyl-tRNA synthetase navigates tRNA on its pathway to the binding site

In the first stage of a diffusion-controlled enzymatic reaction, aminoacyl-tRNA synthetases (aaRSs) interact with cognate tRNAs forming non-specific encounters. The aaRSs catalyzing the same overall aminoacylation reaction vary greatly in subunit organization, structural domain composition and amino acid sequence. The diffusional association of aaRS and tRNA was found to be governed by long-range electrostatic interactions when the homogeneous negative potential of tRNA fits to the patches of positive potential produced by aaRS; one patch for each tRNA substrate molecule. Considering aaRS as a molecule with anisotropic reactivity and on the basis of continuum electrostatics and Smoluchowski's theory, the reaction conditions for tRNA-aaRS diffusional encounters were formulated. The domains, categorized as enzymatically relevant, appeared to be non-essential for field sculpturing at long distances. On the other hand, a set of complementary domains exerts primary control on the aaRS isopotential surface formation. Subdividing the aaRS charged residues into native, conservative and non-conservative subsets, we evaluated the contribution of each group to long-range electrostatic potential. Surprisingly, the electrostatic potential landscapes generated by native and non-conservative subsets are fairly similar, thus suggesting the non-conservative subset is developed specifically for efficient tRNA attraction.     

Structure and activity of an aminoacyl-tRNA synthetase that charges tRNA with nitro-tryptophan

The most divergent of two tryptophanyl tRNA synthetases (TrpRS II) found in Deinococcus radiodurans interacts with a nitric oxide synthase protein that produces 4-nitro-tryptophan (4-NRP). TrpRS II efficiently charges transfer RNA(Trp) with 4-NRP and 5-hydroxy-tryptophan (5-HRP). The crystal structures of TrpRS II bound to tryptophan and 5-HRP reveal residue substitutions that accommodate modified indoles. A class of auxiliary bacterial TrpRSs conserve this capacity to charge tRNA with nonstandard amino acids.     

Discovery of two distinct aminoacyl-tRNA synthetase complexes anchored to the Plasmodium surface tRNA import protein

Aminoacyl-tRNA synthetases (aaRSs) attach amino acids to their cognate transfer RNAs. In eukaryotes, a subset of cytosolic aaRSs is organized into a multisynthetase complex (MSC), along with specialized scaffolding proteins referred to as aaRS-interacting multifunctional proteins (AIMPs). In Plasmodium, the causative agent of malaria, the tRNA import protein (tRip), is a membrane protein that participates in tRNA trafficking; we show that tRip also functions as an AIMP. We identified three aaRSs, the glutamyl-tRNA synthetase (ERS), glutaminyl-tRNA synthetase (QRS), and methionyl-tRNA synthetase (MRS), which were specifically coimmunoprecipitated with tRip in Plasmodium berghei blood stage parasites. All four proteins contain an N-terminal glutathione-S-transferase (GST)–like domain that was demonstrated to be involved in MSC assembly. In contrast to previous studies, further dissection of GST-like interactions identified two exclusive heterotrimeric complexes: the Q-complex (tRip–ERS–QRS) and the M-complex (tRip–ERS–MRS). Gel filtration and light scattering suggest a 2:2:2 stoichiometry for both complexes but with distinct biophysical properties and mutational analysis further revealed that the GST-like domains of QRS and MRS use different strategies to bind ERS. Taken together, our results demonstrate that neither the singular homodimerization of tRip nor its localization in the parasite plasma membrane prevents the formation of MSCs in Plasmodium. Besides, the extracellular localization of the tRNA-binding module of tRip is compensated by the presence of additional tRNA-binding modules fused to MRS and QRS, providing each MSC with two spatially distinct functions: aminoacylation of intraparasitic tRNAs and binding of extracellular tRNAs. This unique host–pathogen interaction is discussed.     

The influence of salt concentration on CD spectra of tRNA and aminoacyl-tRNA synthetase

The CD spectra of serine tRNA or seryl-tRNA synthetase were measured. The [theta] values at 210 nm were minimum at 50mM - 0.2M NaCl, at that concentration the velocity of aminoacylation was maximum. This results suggest that A . U and G . C base pairs loosened. The [theta] values at 200 nm decreased according to the decreasing of salt concentration, suggesting the decomposition of A . U base pairs. The CD spectra of seryl-tRNA synthetase at 210-240 nm were not changed in the range of 10mM-0.3M NaCl but the spectra at 260-290 nm showed minimum in the range between 50mM-0.2M NaCl. These results suggest that the influence of salt concentration on the velocity of aminoacylation depends on both the conformational changes of tRNA and seryl-tRNA synthetase.     

tRNA sulfurtransferase: a member of the aminoacyl-tRNA synthetase complex in rat liver

Transfer RNA sulfurtransferase, tRNA methyltransferase, and aminoacyl-tRNA synthetase activity are associated in a complex in rat liver, which is excluded from Sephadex G-200 columns. The complex can also be isolated by subjecting cell supernatants to further centrifugation at 160,000 x g for 18 hours. The resulting pellet contains 70% of the total sulfurtransferase activity, and a 3-fold increase in specific activity is accomplished through pelleting. The data suggest that the enzymes of tRNA metabolism are organized in a large complex in rat liver.     

Magnesium ion-mediated binding to tRNA by an amino-terminal peptide of a class II tRNA synthetase

Aspartyl-tRNA synthetase is a class II tRNA synthetase and occurs in a multisynthetase complex in mammalian cells. Human Asp-tRNA synthetase contains a short 32-residue amino-terminal extension that can control the release of charged tRNA and its direct transfer to elongation factor 1 alpha; however, whether the extension binds to tRNA directly or interacts with the synthetase active site is not known. Full-length human AspRS, but not amino-terminal 32 residue-deleted, fully active AspRS, was found to bind to noncognate tRNA(fMet) in the presence of Mg(2+). Synthetic amino-terminal peptides bound similarly to tRNA(fMet), whereas little or no binding of polynucleotides, poly(dA-dT), or polyphosphate to the peptides was found. The apparent binding constants to tRNA by the peptide increased with increasing concentrations of Mg(2+), suggesting Mg(2+) mediates the binding as a new mode of RNA.peptide interactions. The binding of tRNA(fMet) to amino-terminal peptides was also observed using fluorescence-labeled tRNAs and circular dichroism. These results suggest that a small peptide can bind to tRNA selectively and that evolution of class II tRNA synthetases may involve structural changes of amino-terminal extensions for enhanced selective binding of tRNA.     

tRNA-dependent aminoacyl-adenylate hydrolysis by a nonediting class I aminoacyl-tRNA synthetase

Glutaminyl-tRNA synthetase generates Gln-tRNA(Gln) 10(7)-fold more efficiently than Glu-tRNA(Gln) and requires tRNA to synthesize the activated aminoacyl adenylate in the first step of the reaction. To examine the role of tRNA in amino acid activation more closely, several assays employing a tRNA analog in which the 2'-OH group at the 3'-terminal A76 nucleotide is replaced with hydrogen (tRNA(2'HGln)) were developed. These experiments revealed a 10(4)-fold reduction in kcat/Km in the presence of the analog, suggesting a direct catalytic role for tRNA in the activation reaction. The catalytic importance of the A76 2'-OH group in aminoacylation mirrors a similar role for this moiety that has recently been demonstrated during peptidyl transfer on the ribosome. Unexpectedly, tracking of Gln-AMP formation utilizing an alpha-32P-labeled ATP substrate in the presence of tRNA(2'HGln) showed that AMP accumulates 5-fold more rapidly than Gln-AMP. A cold-trapping experiment revealed that the nonenzymatic rate of Gln-AMP hydrolysis is too slow to account for the rapid AMP formation; hence, the hydrolysis of Gln-AMP to form glutamine and AMP must be directly catalyzed by the GlnRS x tRNA(2'HGln) complex. This hydrolysis of glutaminyl adenylate represents a novel reaction that is directly analogous to the pre-transfer editing hydrolysis of noncognate aminoacyl adenylates by editing synthetases such as isoleucyl-tRNA synthetase. Because glutaminyl-tRNA synthetase does not possess a spatially separate editing domain, these data demonstrate that a pre-transfer editing-like reaction can occur within the synthetic site of a class I tRNA synthetase.     

tRNA-dependent active site assembly in a class I aminoacyl-tRNA synthetase

The crystal structure of ligand-free E. coli glutaminyl-tRNA synthetase (GlnRS) at 2.4 A resolution shows that substrate binding is essential to construction of a catalytically proficient active site. tRNA binding generates structural changes throughout the enzyme, repositioning key active site peptides that bind glutamine and ATP. The structure gives insight into longstanding questions regarding the tRNA dependence of glutaminyl adenylate formation, the coupling of amino acid and tRNA selectivities, and the roles of specific pathways for transmission of tRNA binding signals to the active site. Comparative analysis of the unliganded and tRNA-bound structures shows, in detail, how flexibility is built into the enzyme architecture and suggests that the induced-fit transitions are a key underlying determinant of both amino acid and tRNA specificity.     

Expanding the genetic code of Caenorhabditis elegans using bacterial aminoacyl-tRNA synthetase/tRNA pairs

The genetic code specifies 20 common amino acids and is largely preserved in both single and multicellular organisms. Unnatural amino acids (Uaas) have been genetically incorporated into proteins by using engineered orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pairs, enabling new research capabilities and precision inaccessible with common amino acids. We show here that Escherichia coli tyrosyl and leucyl amber suppressor tRNA/RS pairs can be evolved to incorporate different Uaas in response to the amber stop codon UAG into various proteins in Caenorhabditis elegans. To accurately report Uaa incorporation in worms, we found that it is crucial to integrate the UAG-containing reporter gene into the genome rather than to express it on an extrachromosomal array from which variable expression can lead to reporter activation independent of the amber-suppressing tRNA/RS. Synthesizing a Uaa in a dipeptide drives Uaa uptake and bioavailability. Uaa incorporation has dosage, temporal, tRNA copy, and temperature dependencies similar to those of endogenous amber suppression. Uaa incorporation efficiency was improved by impairing the nonsense-mediated mRNA decay pathway through knockdown of smg-1. We have generated stable transgenic worms capable of genetically encoding Uaas, enabling Uaa exploitation to address complex biological problems within a metazoan. We anticipate our strategies will be generally extendable to other multicellular organisms.     

Coordination of Mn++ ions at contact sites between tRNA and aminoacyl-tRNA synthetase.

By means of PMR and ESR study the shielding of Mn⁺⁺ ions by aminoacyl-tRNA synthetase has been detected in the aminoacyl-tRNA synthetase - tRNA complex at pH 7.5. At pH 6 this effect was not observed. We propose that ions interact with certain aminoacyl-tRNA synthetase groups protonated when passing to slightly acid pH. The role of Mn⁺⁺ and Mg⁺⁺ ions in the formation of a functionally active complex tRNA-aminoacyl-tRNA synthetase is discussed.     

Crystallization of a tRNA . aminoacyl-tRNA synthetase complex. Characterization and first crystallographic data

A complex formed between the dimeric aspartyl-tRNA synthetase from yeast (Mr congruent to 125,000) and two molecules of its cognate yeast tRNAAsp (Mr = 24,160) was crystallized using ammonium sulfate as the precipitant. The crucial parameter which governs a successful crystallization is the enzyme tRNA stoichiometry. Crystals are only obtained when the starting solution precisely contains two tRNA molecules for one enzyme molecule. It was demonstrated by electrophoresis, biological activity assays, and crystallographic data that the crystals contain the two components in the same two to one stoichiometric ratio. The crystals, of cubic shape with edges up to 0.8 mm, belong to space group 1432. The cell parameter is 354 A and the asymmetric unit contains one particle of complex. The solvent content is about 78%, higher than the values commonly observed. Although particularly soft, the quality of the crystals is suitable for x-ray diffraction studies up to 7-A resolution.     

Amino acid-dependent transfer RNA affinity in a class I aminoacyl-tRNA synthetase

Steady-state and transient kinetic analyses of glutaminyl-tRNA synthetase (GlnRS) reveal that the enzyme discriminates against noncognate glutamate at multiple steps during the overall aminoacylation reaction. A major portion of the selectivity arises in the amino acid activation portion of the reaction, whereas the discrimination in the overall two-step reaction arises from very weak binding of noncognate glutamate. Further transient kinetics experiments showed that tRNA(Gln) binds to GlnRS approximately 60-fold weaker when noncognate glutamate is present and that glutamate reduces the association rate of tRNA with the enzyme by 100-fold. These findings demonstrate that amino acid and tRNA binding are interdependent and reveal an important additional source of specificity in the aminoacylation reaction. Crystal structures of the GlnRS x tRNA complex bound to either amino acid have previously shown that glutamine and glutamate bind in distinct positions in the active site, providing a structural basis for the amino acid-dependent modulation of tRNA affinity. Together with other crystallographic data showing that ligand binding is essential to assembly of the GlnRS active site, these findings suggest a model for specificity generation in which required induced-fit rearrangements are significantly modulated by the identities of the bound substrates.