Ivan Ahel

Trans-editing of mischarged tRNAs.

Aminoacyl-tRNA synthetases (aaRSs) are multidomain proteins that specifically attach amino acids to their cognate tRNAs. Their most conserved, and presumably evolutionarily oldest, domains are the catalytic cores, which activate amino acids and transfer them to the 3′ ends of tRNAs. Additional domains appended to or inserted in the body of aaRSs increase efficiency and specificity of the aminoacylation process, either by providing additional tRNA contacts, or by hydrolyzing noncognate amino acid products (cis-editing). Here, we report specific tRNA-dependent trans-editing by aaRS-like proteins that reciprocate the editing domains of aaRSs, but not the remainder of the corresponding enzyme. A freestanding homologue of the prolyl-tRNA synthetase-editing domain, the PrdX protein from Clostridium sticklandii, efficiently and specifically hydrolyzes Ala-tRNAPro. Similarly, autonomous alanyl-tRNA synthetase-editing domain homologues (AlaX proteins) from Methanosarcina barkeri and Sulfolobus solfataricus hydrolyze Ser-tRNAAla and Gly-tRNAAla substrates. The discovery of autonomous editing proteins efficient in hydrolyzing misacylated products provides a direct link between ancestral aaRSs consisting solely of the catalytic core and extant enzymes to which functionally independent modules are appended.

Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates

Glutamyl-tRNA synthetases (GluRSs) occur in two types, the discriminating and the nondiscriminating enzymes. They differ in their choice of substrates and use either tRNAGlu or both tRNAGlu and tRNAGln. Although most organisms encode only one GluRS, a number of bacteria encode two different GluRS proteins; yet, the tRNA specificity of these enzymes and the reason for such gene duplications are unknown. A database search revealed duplicated GluRS genes in >20 bacterial species, suggesting that this phenomenon is not unusual in the bacterial domain. To determine the tRNA preferences of GluRS, we chose the duplicated enzyme sets from Helicobacter pylori and Acidithiobacillus ferrooxidans. H. pylori contains one tRNAGlu and one tRNAGln species, whereas A. ferrooxidans possesses two of each. We show that the duplicated GluRS proteins are enzyme pairs with complementary tRNA specificities. The H. pylori GluRS1 acylated only tRNAGlu, whereas GluRS2 was specific solely for tRNAGln. The A. ferrooxidans GluRS2 preferentially charged \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{tRNA}}_{{\mathrm{UUG}}}^{{\mathrm{Gln}}}\end{equation*}\end{document}. Conversely, A. ferrooxidans GluRS1 glutamylated both tRNAGlu isoacceptors and the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{tRNA}}_{{\mathrm{CUG}}}^{{\mathrm{Gln}}}\end{equation*}\end{document} species. These three tRNA species have two structural elements in common, the augmented D-helix and a deletion of nucleotide 47. It appears that the discriminating or nondiscriminating natures of different GluRS enzymes have been derived by the coevolution of protein and tRNA structure. The coexistence of the two GluRS enzymes in one organism may lay the groundwork for the acquisition of the canonical glutaminyl-tRNA synthetase by lateral gene transfer from eukaryotes.

Cysteine activation is an inherent in vitro property of prolyl-tRNA synthetases.

Aminoacyl-tRNA synthetases are well known for their remarkable precision in substrate selection during aminoacyl-tRNA formation. Some synthetases enhance the accuracy of this process by editing mechanisms that lead to hydrolysis of incorrectly activated and/or charged amino acids. Prolyl-tRNA synthetases (ProRSs) can be divided into two structurally divergent groups, archaeal-type and bacterial-type enzymes. A striking difference between these groups is the presence of an insertion domain (∼180 amino acids) in the bacterial-type ProRS. Because the archaeal-type ProRS enzymes have been shown to recognize cysteine, we tested selected ProRSs from all three domains of life to determine whether cysteine activation is a general property of ProRS. Here we show that cysteine is activated by recombinant ProRS enzymes from the archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus, from the eukaryote Saccharomyces cerevisiae, and from the bacteria Aquifex aeolicus, Borrelia burgdorferi, Clostridium sticklandii, Cytophaga hutchinsonii, Deinococcus radiodurans, Escherichia coli, Magnetospirillum magnetotacticum, Novosphingobium aromaticivorans, Rhodopseudomonas palustris, and Thermus thermophilus.This non-cognate amino acid was efficiently acylated in vitro onto tRNAPro, and the misacylated Cys-tRNAPro was not edited by ProRS. Therefore, ProRS exhibits a natural level of mischarging that is to date unequalled among the aminoacyl-tRNA synthetases.

Methanocaldococcus jannaschii Prolyl-tRNA Synthetase Charges tRNAPro with Cysteine

Methanocaldococcus jannaschiiprolyl-tRNA synthetase (ProRS) was previously reported to also catalyze the synthesis of cysteinyl-tRNACys(Cys-tRNACys) to make up for the absence of the canonical cysteinyl-tRNA synthetase in this organism (Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H., Ibba, M., and Söll, D. (2000) Science 287, 479–482; Lipman, R. S., Sowers, K. R., and Hou, Y. M. (2000)Biochemistry 39, 7792–7798). Here we show by acid urea gel electrophoresis that pure heterologously expressed recombinantM. jannaschii ProRS misaminoacylates M. jannaschii tRNAPro with cysteine. The enzyme is unable to aminoacylate purified mature M. jannaschiitRNACys with cysteine in contrast to facile aminoacylation of the same tRNA with cysteine by Methanococcus maripaludiscysteinyl-tRNA synthetase. Although M. jannaschii ProRS catalyzes the synthesis of Cys-tRNAPro readily, the enzyme is unable to edit this misaminoacylated tRNA. We discuss the implications of these results on the in vivo activity of the M. jannaschii ProRS and on the nature of the enzyme involved in the synthesis of Cys-tRNACys in M. jannaschii.

Cys-tRNACys formation and cysteine biosynthesis in methanogenic archaea: two faces of the same problem?

Abstract.Aminoacyl-tRNA (transfer RNA) synthetases are essential components of the cellular translation machinery as they provide the ribosome with aminoacyl-tRNAs. Aminoacyl-tRNA synthesis is generally well understood. However, the mechanism of Cys-tRNACys formation in three methanogenic archaea (Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus and Methanopyrus kandleri) is still unknown, since no recognizable gene for a canonical cysteinyl-tRNA synthetase could be identified in the genome sequences of these organisms. Here we review the different routes recently proposed for Cys-tRNACys formation and discuss its possible link with cysteine biosynthesis in these methanogenic archaea.

Cysteinyl-tRNA formation and prolyl-tRNA synthetase.

Aminoacyl‐tRNA (AA‐tRNA) formation is a key step in protein biosynthesis. This reaction is catalyzed with remarkable accuracy by the AA‐tRNA synthetases, a family of 20 evolutionarily conserved enzymes. The lack of cysteinyl‐tRNA (Cys‐tRNA) synthetase in some archaea gave rise to the discovery of the archaeal prolyl‐tRNA (Pro‐tRNA) synthetase, an enzyme capable of synthesizing Pro‐tRNA and Cys‐tRNA. Here we review our current knowledge of this fascinating process.

Aminoacyl-tRNA formation in the extreme thermophile Thermus thermophilus.

Abstract. Thermophilic organisms must be capable of accurate translation at temperatures in which the individual components of the translation machinery and also specific amino acids are particularly sensitive. Thermus thermophilus is a good model organism for studies of thermophilic translation because many of the components in this process have undergone structural and biochemical characterization. We have focused on the pathways of aminoacyl-tRNA synthesis for glutamine, asparagine, proline, and cysteine. We show that the T. thermophilus prolyl-tRNA synthetase (ProRS) exhibits cysteinyl-tRNA synthetase (CysRS) activity although the organism also encodes a canonical CysRS. The ProRS requires tRNA for cysteine activation, as is known for the characterized archaeal prolyl-cysteinyl-tRNA synthetase (ProCysRS) enzymes. The heterotrimeric T. thermophilus aspartyl-tRNAAsn amidotransferase can form Gln-tRNA in addition to Asn-tRNA; however, a 13-amino-acid C-terminal truncation of the holoenzyme A subunit is deficient in both activities when assayed with homologous substrates. A survey of codon usage in completed prokaryotic genomes identified a higher Glu:Gln ratio in proteins of thermophiles compared to mesophiles.