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Coevolution of an Aminoacyl-Trna Synthetase with Its Trna Substrates

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Coevolution of an Aminoacyl-Trna Synthetase with Its Trna Substrates Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates Juan C. Salazar*†, Ivan Ahel*†, Omar Orellana*‡, Debra Tumbula-Hansen*, Robert Krieger*, Lacy Daniels§, and Dieter So¨ ll*¶ʈ Departments of *Molecular Biophysics and Biochemistry and ¶Chemistry, Yale University, New Haven, CT 06520-8114; ‡Programa de Biologı´aCelular y Molecular, Instituto de Ciencias Biome´dicas, Facultad de Medicina, Universidad de Chile, Casilla 70086 Santiago 7, Chile; and §Department of Microbiology and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, IA 52242 Contributed by Dieter So¨ll, September 23, 2003 Glutamyl-tRNA synthetases (GluRSs) occur in two types, the dis- In bacteria, the ND Bacillus subtilis GluRS has been exten- criminating and the nondiscriminating enzymes. They differ in sively studied (9). Due to the lack of a canonical glutaminyl- their choice of substrates and use either tRNAGlu or both tRNAGlu tRNA synthetase (GlnRS) in this organism, the ND-GluRS is an and tRNAGln. Although most organisms encode only one GluRS, a essential enzyme in Gln-tRNA formation as it generates Glu- number of bacteria encode two different GluRS proteins; yet, the tRNAGln. This product is then converted to Gln-tRNAGln by tRNA specificity of these enzymes and the reason for such gene Glu-tRNAGln amidotransferase (10–12). However, the mecha- duplications are unknown. A database search revealed duplicated nism of how the ND-GluRS recognizes two different tRNA GluRS genes in >20 bacterial species, suggesting that this phe- substrates is not known, and the tRNA identity set for such an nomenon is not unusual in the bacterial domain. To determine the enzyme has never been determined. In contrast, organisms with tRNA preferences of GluRS, we chose the duplicated enzyme sets a canonical GlnRS enzyme possess a discriminating GluRS Glu from Helicobacter pylori and Acidithiobacillus ferrooxidans. H. (D-GluRS), which recognizes only tRNA . The tRNA identity pylori contains one tRNAGlu and one tRNAGln species, whereas A. elements of a D-GluRS enzyme were reported for E. coli (13). ferrooxidans possesses two of each. We show that the duplicated Major identity determinants for this enzyme are located in the GluRS proteins are enzyme pairs with complementary tRNA spec- augmented D-helix (indicated in Fig. 1), which is formed by ificities. The H. pylori GluRS1 acylated only tRNAGlu, whereas nucleotides in the D-stem and D-loop, as well as the bases GluRS2 was specific solely for tRNAGln. The A. ferrooxidans GluRS2 connecting the acceptor helix and D-stem, and some bases in the Gln extra arm (13). In vitro mutagenesis of Thermus thermophilus preferentially charged tRNAUUG. Conversely, A. ferrooxidans GluRS1 glutamylated both tRNAGlu isoacceptors and the tRNAGln D-GluRS expanded its tRNA specificity to the recognition of CUG tRNAGlu with a glutamine-specific anticodon (14). However, this species. These three tRNA species have two structural elements in work did not afford real insight into a ND-GluRS, because the common, the augmented D-helix and a deletion of nucleotide 47. tRNA specificity was not changed. It appears that the discriminating or nondiscriminating natures of In addition to the lack of some synthetases (see above), whole different GluRS enzymes have been derived by the coevolution of genome analysis also revealed several organisms containing protein and tRNA structure. The coexistence of the two GluRS more than one AARS for certain amino acids. In many cases, the enzymes in one organism may lay the groundwork for the acqui- function of such a redundancy is unknown; however, in some sition of the canonical glutaminyl-tRNA synthetase by lateral gene organisms, the roles of the duplicated AARSs have been ex- transfer from eukaryotes. plained (15). A second aspartyl-tRNA synthetase in Deinococcus radiodurans, for instance, is shown to be involved in tRNA- aithful protein biosynthesis relies on the correct attachment dependent asparagine biosynthesis (7). In E. coli, two genes for Fof amino acids to their corresponding (cognate) tRNAs lysyl-tRNA synthetase (LysRS) exist; one LysRS gene (lysS)is catalyzed by the aminoacyl-tRNA synthetases (AARSs) (1). constitutively expressed, and the other one is heat-inducible Given the presence of 20 canonical amino acids, one would (lysU) (16, 17). Similarly, two paralogous gltX genes (encoding expect each organism to contain at least 20 AARSs to ensure GluRS) exist in some proteobacteria and a scattering of other high accuracy during aminoacylation of the complete set of bacterial groups (18); however, the function of this duplication tRNAs. However, this is true only for eukarya and some bacteria. is not clear. There are indications that the two GluRSs in Most of the other bacterial organisms (2, 3), all known archaea Helicobacter pylori preferentially glutamylate either tRNAGlu or Gln (4, 5), and eukaryotic organelles (6) lack the AARS specific for tRNA .** To investigate further the duplicated gltX genes, we glutamine. In addition, a canonical asparaginyl-tRNA synthetase chose the Acidithiobacillus ferrooxidans and H. pylori systems and is missing from many archaeal and bacterial genomes (7), examined the substrate preferences of the two GluRS enzymes whereas a canonical cysteinyl-tRNA synthetase is not distin- that coexist in these organisms. guishable in some methanogenic archaea (8). Materials and Methods In addition to direct acylation of the cognate tRNA by Oligonucleotides, DNA Sequencing, and Radiochemicals. AARSs, an indirect pathway was found to be responsible for the Oligonucle- otides were synthesized, and DNAs were sequenced by the Keck formation of both Gln-tRNAGln and Asn-tRNAAsn. In these Foundation Biotechnology Resource Laboratory at Yale Uni- cases, formation of the mischarged product (Glu-tRNAGln and versity. Uniformly labeled [14C]Glu (254 mCi͞mmol) and BIOCHEMISTRY Asp-tRNAAsn, respectively) is a required intermediate in the synthesis of the cognate amino acid–tRNA pairs. To accomplish Gln this, the indirect pathway to Gln-tRNA formation uses a Abbreviations: AARS, aminoacyl-tRNA synthetase; ND, nondiscriminating; GluRS, glu- nondiscriminating (ND) form of glutamyl-tRNA synthetase tamyl-tRNA synthetase; GlnRS, glutaminyl-tRNA synthetase. (GluRS) that is able to efficiently glutamylate both tRNAGlu and †J.C.S. and I.A. contributed equally to this work. Gln tRNA (9). In the next step, a tRNA-dependent amidotrans- ʈTo whom correspondence should be addressed. E-mail: [email protected]. Gln Gln ferase, Glu-tRNA amidotransferase, amidates Glu-tRNA **Hendrickson, T. L., Skouloubris, S., de Reuse, H. & Labigne, A. (2002) Abstr. Papers Am. to the correctly charged Gln-tRNAGln, which is then used as a Chem. Soc. 224, 121. substrate for protein synthesis (reviewed in ref. 10). © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.1936123100 PNAS ͉ November 25, 2003 ͉ vol. 100 ͉ no. 24 ͉ 13863–13868 Downloaded by guest on October 1, 2021 ligation into the pKK223-3 vector (Amersham Biosciences) and digested with EcoRI and HindIII enzymes, generating the plasmids pJSQUUG and pJSQCUG. These were transformed into E. coli DH5␣. Purification of unfractionated tRNA containing the spec- ified tRNA gene transcript was carried out as described (20). Comparison of aminoacylation reactions by using E. coli GlnRS and unfractionated tRNA obtained from the cells, transformed with pJSQUUG, pJSQCUG, or empty vector, showed that the A. Gln Gln ferrooxidans tRNAUUG and tRNACUG isoacceptors comprised be- tween 10% and 20% of the total tRNA. To obtain mature unfractionated tRNA from both organisms, H. pylori cells (strain 43526) were grown (21), and the unfraction- ated tRNA was prepared as described (20). A. ferrooxidans (strain 19859) was grown in a bioreactor in 9-K medium [0.4 g͞liter ͞ ͞ ͞ (NH4)2SO4,0.4g l MgSO4(7H2O), 0.056 g lK2HPO4(3H2O) 5g/l FeSO4(7H2O) adjusted to pH 1.6 with concentrated H2SO4, and sterilized by filtration. The ferrous ion was regenerated by 0.5 A of current]. Total tRNA extracted from 0.5 g of A. ferrooxidans cells was purified by the Qiagen RNA͞DNA kit as described by the manufacturer (Qiagen, Valencia, CA). Purification of tRNAGln and tRNAGlu by Affinity Chromatography. The Gln Gln A. ferrooxidans tRNAUUG and tRNACUG (see above) and E. coli tRNAGln and tRNAGlu were purified from unfractionated tRNA by affinity chromatography on immobilized T. thermophilus EF-Tu (22) with the following specifications. Unfractionated E. coli tRNA was aminoacylated with E. coli GlnRS or GluRS. The Gln-tRNAGln or Glu-tRNAGlu was separated from the un- Fig. 1. Cloverleaf representation of A. ferrooxidans tRNAGlu and tRNAGln charged tRNA by the formation of a ternary complex with the species. The bases that form part of the augmented D-helix (13), in both EF-Tu-GTP immobilized on a Ni-NTA-agarose column. After Glu Gln Gln Glu isoacceptors of tRNA and in tRNACUG, are shown in white letters and black the elution of Gln-tRNA or Glu-tRNA , the tRNA was shadow. deacylated for 15 min at 65°C in 0.1 mM borate-KOH, pH 9.0. The pure tRNAGln was not contaminated with tRNAGlu and vice 14 ͞ ␥ 32 ͞ versa, as judged by aminoacylation with pure E. coli GluRS or [ C]Gln (244 mCi mmol) and [ - P]ATP (6,000 Ci mmol) GlnRS, respectively. were from Amersham Pharmacia Biosciences. Aminoacylation Assays with GluRS1 and -RS2. In vitro acylations with Cloning of gltX Genes from H. pylori and A. ferrooxidans. The H. H. pylori and A. ferrooxidans GluRS enzymes was carried out at pylori genes (HP0476, gltX1; HP0643, gltX2) were obtained by 37°Cinan80-␮l reaction mixture containing 100 mM Hepes- PCR from the genomic DNA (strain 26695) by using the Expand KOH, pH 7.2; 30 mM KCl; 12 mM MgCl2; 5 mM ATP; 2 mM High Fidelity PCR System (Roche Molecular Biochemicals). DTT; 25 ␮M[14C]Glu plus 75 ␮M unlabeled glutamate and Oligonucleotide primers introduced an NdeI site at the 5Ј end ␮ ␮ Ј 40–60 g of unfractionated tRNA or 0.7–1.3 g of pure tRNA.
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