Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases (original) (raw)
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Biochemistry, 1997
Primordial aminoacyl-tRNA synthetases (aaRSs) based on the Rossman nucleotide binding fold of class I enzymes or the seven-stranded antiparallel -sheet fold of class II enzymes have been proposed to predate the contemporary aaRS. As part of an inquiry into class II aaRS evolution, the individual domains of the homodimeric Escherichia coli histidyl-tRNA synthetase (HisRS) were separately expressed and purified to determine their individual contributions to catalysis. A 320-residue fragment (N cat HisRS) truncated immediately following motif 3 catalyzes both the specific aminoacylation of tRNA and pyrophosphate exchange, albeit less efficiently than the full-length enzyme. N cat HisRS showed no mischarging of noncognate tRNAs but exhibited reduced selectivity for the C73 discriminator base, a principal aminoacylation determinant for histidine tRNAs. Size exclusion chromatography showed that N cat HisRS is monomeric, indicating that the C-terminal domain is essential for maintaining the dimeric structure of the enzyme. The stably folded C-terminal domain (C ter HisRS) was inactive for both reactions and did not enhance the activity of N cat HisRS when added in trans. The fusion of one or more accessory domains to a primordial catalytic domain may therefore have been a critical evolutionary step by which aminoacyl-tRNA synthetases acquired increased catalytic efficiency and substrate specificity. X Abstract published in AdVance ACS Abstracts, March 15, 1997. 1 Abbreviations: aaRS, aminoacyl-tRNA synthetase; HPLC, highpressure liquid chromatography; IPTG, isopropyl 1-thio--D-galactopyranoside; HEPES, N-(2-hydroxyethyl)piperazine-N ′-2-ethanesulfonic acid; Ni-NTA, nickel nitrilotriacetic acid; NOESY, nuclear Overhauser enhancement spectroscopy; PCR, polymerase chain reaction. Following the standard convention, aminoacyl-tRNA synthetases are referred to by the three-letter code for their amino acid substrate, followed by the suffix RS.
Biochemistry, 1994
A core of eight beta-strands and three alpha-helices was recently predicted for the active site domain of Escherichia coli alanyl-tRNA synthetase, an enzyme of unknown structure [Ribas de Pouplana, L1., Buechter, D. D., Davis, M. W., & Schimmel, P. (1993) Protein Sci. 2, 2259-2262; Shi, J.-P., Musier-Forsyth, K., & Schimmel, P. (1994) Biochemistry 26, 5312-5318]. A critical part of this predicted structure is two antiparallel beta-strands and an intervening loop that make up the second of three highly degenerate sequence motifs that are characteristic of the class II aminoacyl-tRNA synthetases. We present here an in vivo and in vitro analysis of 21 rationally designed mutations in the predicted 34-amino acid motif 2 of E. coli alanyl-tRNA synthetase. Although this motif in E. coli alanyl-tRNA synthetase is of a different size than and has only two sequence identities with the analogous motif in yeast aspartyl- and Thermus thermophilus seryl-tRNA synthetases, whose structures are known, the functional consequences of the mutations are explainable in terms of those structures. In particular, the analysis demonstrates the importance of the predicted motif 2 in adenylate formation, distinguishes between two similar, but distinct, predicted models for this motif, and distinguishes between the functional importance of two adjacent phenylalanines in a way that strongly supports the predicted structure. The results suggest that similar analyses will be generally useful in testing models for active site regions of other class II aminoacyl-tRNA synthetases of unknown structure.
The evolution of Class II Aminoacyl-tRNA synthetases and the first code
FEBS Letters, 2015
Class II Aminoacyl-tRNA synthetases are a set of very ancient multi domain proteins. The evolution of the catalytic domain of Class II synthetases can be reconstructed from three peptidyl-hairpins. Further evolution from this primordial catalytic core leads to a split of the Class II synthetases into two divisions potentially associated with the operational code. The earliest form of this code likely coded predominantly Glycine (Gly), Proline (Pro), Alanine (Ala) and ''Lysine"/Aspartic acid (Lys/Asp). There is a paradox in these synthetases beginning with a hairpin structure before the Genetic Code existed. A resolution is found in the suggestion that the primordial Aminoacyl synthetases formed in a transition from a Thioester world to a Phosphate ester world.
Coding of Class I and II Aminoacyl-tRNA Synthetases
Advances in Experimental Medicine and Biology, 2017
The aminoacyl-tRNA synthetases and their cognate transfer RNAs translate the universal genetic code. The twenty canonical amino acids are sufficiently diverse to create a selective advantage for dividing amino acid activation between two distinct, apparently unrelated superfamilies of synthetases, Class I amino acids being generally larger and less polar, Class II amino acids smaller and more polar. Biochemical, bioinformatic, and protein engineering experiments support the hypothesis that the two Classes descended from opposite strands of the same ancestral gene. Parallel experimental deconstructions of Class I and II synthetases reveal parallel losses in catalytic proficiency at two novel modular levels-protozymes and Urzymes-associated with the evolution of catalytic activity. Bi-directional coding supports an important unification of the proteome; affords a genetic relatedness metric-middle base-pairing frequencies in sense/ antisense alignments-that probes more deeply into the evolutionary history of translation than do single multiple sequence alignments; and has facilitated the analysis of hitherto unknown coding relationships in tRNA sequences. Reconstruction of native synthetases by modular thermodynamic cycles facilitated by domain engineering emphasizes the subtlety associated with achieving high specificity, shedding new light on allosteric relationships in contemporary synthetases. Synthetase Urzyme structural biology suggests that they are catalytically active molten globules, broadening the potential manifold of polypeptide catalysts accessible to primitive genetic coding and motivating revisions of the origins of catalysis. Finally, bi-directional genetic coding of some of the oldest genes in the proteome places major limitations on the likelihood that any RNA World preceded the origins of coded proteins.
Sequence similarities among the family of aminoacyl-tRNA synthetases
Biochimie, 1986
Recent affinity labeling studies have led to the identification of lysine residues at the CCA binding site of tRNA in Escherichia coli methionyl-and tyrosyl-tRNA synthetases. The comparison of the labeled peptides to the known primary structures of the aminoacyl-tRNA synthetases reveals new sequence similarities among this family of enzymes. These similarities include a 'constant' lysine residue whose functional significance is discussed. Moreover, a systematic computer analysis was conducted to search for similarities between the aminoacyl-tRNA synthetases taken as pairs.
Selection of a 'minimal' glutaminyl-tRNA synthetase and the evolution of class I synthetases
The EMBO journal, 1993
The evolution of the aminoacyl-tRNA synthetases is intriguing in light of their elaborate relationship with tRNAs and their significance in the decoding process. Based on sequence motifs and structure determination, these enzymes have been assigned to two classes. The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS), a class I enzyme, complexed to tRNA(Gln) and ATP has been described. It is shown here that a 'minimal' GlnRS, i.e. a GlnRS from which domains interacting with the acceptor-end and the anticodon of the tRNA have been deleted, has enzymatic activity and can charge a tRNA(Tyr)-derived amber suppressor (supF) with glutamine. The catalytic core of GlnRS, which is structurally conserved in other class I synthetases, is therefore sufficient for the aminoacylation of tRNA substrates. Some of these truncated enzymes have lost their ability to discriminate against non-cognate tRNAs, implying a more specific role of the acceptor-end-binding domain i...