Rebecca W. Alexander

Experimental and computational determination of tRNA dynamics

As the molecular representation of the genetic code, tRNA plays a central role in the translational machinery where it interacts with several proteins and other RNAs during the course of protein synthesis. These interactions exploit the dynamic flexibility of tRNA. In this minireview, we discuss the effects of modified bases, ions, and proteins on tRNA structure and dynamics and the challenges of observing its motions over the cycle of translation.

Misacylation of specific nonmethionyl tRNAs by a bacterial methionyl-tRNA synthetase

Aminoacyl-tRNA synthetases perform a critical step in translation by aminoacylating tRNAs with their cognate amino acids. Although high fidelity of aminoacyl-tRNA synthetases is often thought to be essential for cell biology, recent studies indicate that cells tolerate and may even benefit from tRNA misacylation under certain conditions. For example, mammalian cells selectively induce mismethionylation of nonmethionyl tRNAs, and this type of misacylation contributes to a cell’s response to oxidative stress. However, the enzyme responsible for tRNA mismethionylation and the mechanism by which specific tRNAs are mismethionylated have not been elucidated. Here we show by tRNA microarrays and filter retention that the methionyl-tRNA synthetase enzyme from Escherichia coli (EcMRS) is sufficient to mismethionylate two tRNA species, and , indicating that tRNA mismethionylation is also present in the bacterial domain of life. We demonstrate that the anticodon nucleotides of these misacylated tRNAs play a critical role in conferring mismethionylation identity. We also show that a certain low level of mismethionylation is maintained for these tRNAs, suggesting that mismethionylation levels may have evolved to confer benefits to the cell while still preserving sufficient translational fidelity to ensure cell viability. EcMRS mutants show distinct effects on mismethionylation, indicating that many regions in this synthetase enzyme influence mismethionylation. Our results show that tRNA mismethionylation can be carried out by a single protein enzyme, mismethionylation also requires identity elements in the tRNA, and EcMRS has a defined structure-function relationship for tRNA mismethionylation.

Using molecular dynamics to map interaction networks in an aminoacyl‐tRNA synthetase

Long‐range functional communication is a hallmark of many enzymes that display allostery, or action‐at‐a‐distance. Many aminoacyl‐tRNA synthetases can be considered allosteric, in that their trinucleotide anticodons bind the enzyme at a site removed from their catalytic domains. Such is the case with E. coli methionyl‐tRNA synthase (MetRS), which recognizes its cognate anticodon using a conserved tryptophan residue 50 Å away from the site of tRNA aminoacylation. The lack of details regarding how MetRS and tRNAMet interact has limited efforts to deconvolute the long‐range communication that occurs in this system. We have used molecular dynamics simulations to evaluate the mobility of wild‐type MetRS and a Trp‐461 variant shown previously by experiment to be deficient in tRNA aminoacylation. The simulations reveal that MetRS has significant mobility, particularly at structural motifs known to be involved in catalysis. Correlated motions are observed between residues in distant structural motifs, including the active site, zinc binding motif, and anticodon binding domain. Both mobility and correlated motions decrease significantly but not uniformly upon substitution at Trp‐461. Mobility of some residues is essentially abolished upon removal of Trp‐461, despite being tens of Ångstroms away from the site of mutation and solvent exposed. This conserved residue does not simply participate in anticodon binding, as demonstrated experimentally, but appears to mediate the proteins distribution of structural ensembles. Finally, simulations of MetRS indicate that the ligand‐free protein samples conformations similar to those observed in crystal structures with substrates and substrate analogs bound. Thus, there are low energetic barriers for MetRS to achieve the substrate‐bound conformations previously determined by structural methods. Proteins 2007.

Peptide synthesis through evolution.

Ribosome-catalyzed peptide bond formation is a crucial function of all organisms. The ribosome is a ribonucleoprotein particle, with both RNA and protein components necessary for the various steps leading to protein biosynthesis. Evolutionary theory predicts an early environment devoid of complex biomolecules, and prebiotic peptide synthesis would have started in a simple way. A fundamental question regarding peptide synthesis is how the current ribosome-catalyzed reaction evolved from a primitive system. Here we look at both prebiotic and modern mechanisms of peptide bond formation and discuss recent experiments that aim to connect these activities. In particular, RNA can facilitate peptide bond formation by providing a template for activated amino acids to react and can catalyze a variety of functions that would have been necessary in a pre-protein world. Therefore, RNA may have facilitated the emergence of the current protein world from an RNA or even prebiotic world.

Role for a Conserved Structural Motif in Assembly of a Class I Aminoacyl-tRNA Synthetase Active Site

The catalytic domains of class I aminoacyl-tRNA synthetases are built around a conserved Rossmann nucleotide binding fold, with additional polypeptide domains responsible for tRNA binding or hydrolytic editing of misacylated substrates. Structural comparisons identified a conserved motif bridging the catalytic and anticodon binding domains of class Ia and Ib enzymes. This stem contact fold (SCF) has been proposed to globally orient each enzymes cognate tRNA by interacting with the inner corner of the L-shaped tRNA. Despite the structural similarity of the SCF among class Ia/Ib enzymes, the sequence conservation is low. We replaced amino acids of the MetRS SCF with portions of the structurally similar glutaminyl-tRNA synthetase (GlnRS) motif or with alanine residues. Chimeric variants retained significant tRNA methionylation activity, indicating that structural integrity of the helix-turn-strand-helix motif contributes more to tRNA aminoacylation than does amino acid identity. In contrast, chimeras were significantly reduced in methionyl adenylate synthesis, suggesting a role for the SCF in formation of a structured active site domain. A highly conserved aspartic acid within the MetRS SCF is proposed to make an electrostatic interaction with an active site lysine; these residues were replaced with alanines or conservative substitutions. Both methionyl adenylate formation and methionine transfer were impaired, and activity was not significantly recovered by making the compensatory double substitution.

Mitochondrial aminoacyl-tRNA synthetase single-nucleotide polymorphisms that lead to defects in refolding but not aminoacylation.

Defects in organellar translation are the underlying cause of a number of mitochondrial diseases, including diabetes, deafness, encephalopathy, and other mitochondrial myopathies. The most common causes of these diseases are mutations in mitochondria-encoded tRNAs. It has recently become apparent that mutations in nuclear-encoded components of the mitochondrial translation machinery, such as aminoacyl-tRNA synthetases (aaRSs), can also lead to disease. In some cases, mutations can be directly linked to losses in enzymatic activity; however, for many, their effect is unknown. To investigate how aaRS mutations impact function without changing enzymatic activity, we chose nonsynonymous single-nucleotide polymorphisms (nsSNPs) that encode residues distal from the active site of human mitochondrial phenylalanyl-tRNA synthetase. The phenylalanyl-tRNA synthetase variants S57C and N280S both displayed wild-type aminoacylation activity and stability with respect to their free energies of unfolding, but were less stable at low pH. Mitochondrial proteins undergo partial unfolding/refolding during import, and both S57C and N280S variants retained less activity than wild type after refolding, consistent with their reduced stability at low pH. To examine possible defects in protein folding in other aaRS nsSNPs, we compared the refolding of the human mitochondrial leucyl-tRNA synthetase variant H324Q to that of wild type. The H324Q variant had normal activity prior to unfolding, but displayed a refolding defect resulting in reduced aminoacylation compared to wild type after renaturation. These data show that nsSNPs can impact mitochondrial translation by changing a biophysical property of a protein (in this case refolding) without affecting the corresponding enzymatic activity.

An operational RNA code for faithful assignment of AUG triplets to methionine.

The assignment of AUG codons to methionine remains a central question of the evolution of the genetic code. We have unveiled a strategy for the discrimination among tRNAs containing CAU (AUG-decoding) anticodons. Mycoplasma penetrans methionyl-tRNA synthetase can directly differentiate between tRNA(Ile)(CAU) and tRNA(Met)(CAU) transcripts (a recognition normally achieved through the modification of anticodon bases). This discrimination mechanism is based only on interactions with the acceptor stems of tRNA(Ile)(CAU) and tRNA(Met)(CAU). Thus, in certain species, the fidelity of translation of methionine codons requires a discrimination mechanism that is independent of the information contained in the anticodon.

Teasing apart the Taxol pathway

The diterpenoid Taxol (paclitaxel) from the yew (Taxus) species has been used successfully in the treatment of ovarian, breast and lung cancers, as well as of Kaposis sarcoma. The increased demand for Taxol, coupled with its limited availability from the protected Pacific yew, has had researchers scrambling for alternate sources, including synthetic and semi-synthetic pathways. Its structural complexity, however, has precluded a total synthesis suitable for large-scale production. The biosynthetic route to Taxol is no less intricate than that accomplished in the laboratory: there are a dozen enzymatic steps, including five acyltransferase reactions. Croteau and co-workers at Washington State University have already identified several enzymes in the Taxol biosynthetic pathway, including the first and third acyltransferases. Based on the abundance of naturally occurring taxoids, benzoylation at the taxane C2α-hydroxyl position was expected to be the second acylation step. Now, Walker and Croteau report the cloning from Taxus cuspidata of taxane 2α-O-benzoyltransferase, the enzyme responsible for this acylation reaction 1xTaxol biosynthesis: molecular cloning of a benzoyl-CoA:taxane 2α-O-benzoyltransferase cDNA from Taxus and functional expression in Escherichia coli. Walker, K and Croteau, R. Proc. Natl. Acad. Sci. USA. 2000; 97: 13591–13596Crossref | PubMed | Scopus (101)See all References.The researchers tricked the plant cells into producing more Taxol (and therefore more metabolic enzymes) by incubation with methyl jasmonate, a plant signaling molecule involved in environmental stress responses. Several potential clones were isolated using transacylase-specific primers, and these clones exhibited high (64–72%) sequence similarity to previously identified enzymes in the family. Putative constructs were expressed in Escherichia coli and isolated, then evaluated for their ability to use radiolabeled benzoyl-CoA in a transferase reaction. The natural diterpenoid substrate was not available in sufficient quantities to assay the expressed protein, so 2-debenzoyl-7,13-diacetylbaccatin III was prepared semisynthetically. Upon incubation with the soluble enzyme fraction, a biosynthetic product corresponding to authentic 7,13-diacetylbaccatinIII was identified. Product formation was regioselective for the 2α-hydroxyl position and dependent on a high level of ring substitution. The functional benzoyltransferase is a 440-residue protein with over 60% sequence identity to the other two acyltransferases in the Taxol pathway, and contains the previously identified HXXXDG motif that might contribute to acyl group transfer.Taxol achieves its anticancer activity by a novel mechanism: it promotes the assembly of tubulin into microtubules, thereby disrupting mitosis. Taxol resistance is already a problem in several tumor lines, and seems due in part to changes in post-translational modifications of tubulin. Structure–function studies of Taxol have suggested that the C2 benzoate moiety is necessary for tubulin stabilization, and that substitutions at this position could yield taxol derivatives with enhanced potency. Taxane 2α-O-benzoyltransferase might provide an enzymatic means for enhanced production of Taxol and new anticancer drugs derived from this remarkable plant product.

Evidence for Late Resolution of the AUX Codon Box in Evolution

Background: Protein biosynthesis requires accurate tRNA aminoacylation. Results: Bacterial methionyl-tRNA synthetases (MRSs) vary in their ability to reject near-cognate tRNAIle transcripts containing the methionine-specifying CAU anticodon. Conclusion: Given the degree of near-cognate discrimination among bacterial MRSs, aspects of genetic code accuracy likely were fixed relatively late in evolution. Significance: This varied discrimination may reflect differing cellular needs for translational accuracy versus plasticity. Recognition strategies for tRNA aminoacylation are ancient and highly conserved, having been selected very early in the evolution of the genetic code. In most cases, the trinucleotide anticodons of tRNA are important identity determinants for aminoacylation by cognate aminoacyl-tRNA synthetases. However, a degree of ambiguity exists in the recognition of certain tRNAIle isoacceptors that are initially transcribed with the methionine-specifying CAU anticodon. In most organisms, the C34 wobble position in these tRNAIle precursors is rapidly modified to lysidine to prevent recognition by methionyl-tRNA synthetase (MRS) and production of a chimeric Met-tRNAIle that would compromise translational fidelity. In certain bacteria, however, lysidine modification is not required for MRS rejection, indicating that this recognition strategy is not universally conserved and may be relatively recent. To explore the actual distribution of lysidine-dependent tRNAIle rejection by MRS, we have investigated the ability of bacterial MRSs from different clades to differentiate cognate tRNACAUMet from near-cognate tRNACAUIle. Discrimination abilities vary greatly and appear unrelated to phylogenetic or structural features of the enzymes or sequence determinants of the tRNA. Our data indicate that tRNAIle identity elements were established late and independently in different bacterial groups. We propose that the observed variation in MRS discrimination ability reflects differences in the evolution of genetic code machineries of emerging bacterial clades.

Effect of a Domain-Spanning Disulfide on Aminoacyl-tRNA Synthetase Activity

Enzymes regulated by allostery undergo conformational rearrangement upon binding effector molecules. For modular proteins, a flexible interface may mediate reorientation of the protein domains and transmit binding events to activate catalysis at a distance. Aminoacyl-tRNA synthetases (aaRSs) that use tRNA anticodons as identity elements can be considered allosteric enzymes in which aminoacylation of the tRNA acceptor stem is enhanced upon anticodon binding. We reasoned that anticodon-triggered conformational change might be restricted upon introduction of a disulfide linkage near the core of an aaRS. Here we show that a double cysteine mutation engineered at the Escherichia coli MetRS domain interface spontaneously generates a disulfide linkage. This disulfide clamp has no effect on methionyl adenylate formation but reduces the level of tRNA(Met) aminoacylation approximately 2-fold. Activity is restored upon chemical reduction of the disulfide, demonstrating that E. coli MetRS requires a flexible interface domain for full catalytic efficiency.