Activation of the pyrrolysine suppressor tRNA requires formation of a ternary complex with class I and class II lysyl-tRNA synthetases

Monomethylamine methyltransferase of the archaeon Methanosarcina barkeri contains a rare amino acid, pyrrolysine, encoded by the termination codon UAG. Translation of this UAG requires the aminoacylation of the corresponding amber suppressor tRNAPyl. Previous studies reported that tRNAPyl could be aminoacylated by the synthetase-like protein PylS. We now show that tRNAPyl is efficiently aminoacylated in the presence of both the class I LysRS and class II LysRS of M. barkeri, but not by either enzyme acting alone or by PylS. In vitro studies show that both the class I and II LysRS enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Structural modeling and selective inhibition experiments indicate that the class I and II LysRSs form a ternary complex with tRNAPyl, with the aminoacylation activity residing in the class II enzyme.     

Functional annotation of class I lysyl-tRNA synthetase phylogeny indicates a limited role for gene transfer

Functional and comparative genomic studies have previously shown that the essential protein lysyl-tRNA synthetase (LysRS) exists in two unrelated forms. Most prokaryotes and all eukaryotes contain a class II LysRS, whereas most archaea and a few bacteria contain a less common class I LysRS. In bacteria the class I LysRS is only found in the alpha-proteobacteria and a scattering of other groups, including the spirochetes, while the class I protein is by far the most common form of LysRS in archaea. To investigate this unusual distribution we functionally annotated a representative phylogenetic sampling of LysRS proteins. Class I LysRS proteins from a variety of bacteria and archaea were characterized in vitro by their ability to recognize Escherichia coli tRNA(Lys) anticodon mutants. Class I LysRS proteins were found to fall into two distinct groups, those that preferentially recognize the third anticodon nucleotide of tRNA(Lys) (U36) and those that recognize both the second and third positions (U35 and U36). Strong recognition of U35 and U36 was confined to the pyrococcus-spirochete grouping within the archaeal branch of the class I LysRS phylogenetic tree, while U36 recognition was seen in other archaea and an example from the alpha-proteobacteria. Together with the corresponding phylogenetic relationships, these results suggest that despite its comparative rarity the distribution of class I LysRS conforms to the canonical archaeal-bacterial division. The only exception, suggested from both functional and phylogenetic data, appears to be the horizontal transfer of class I LysRS from a pyrococcal progenitor to a limited number of bacteria.     

Discrimination of cognate and noncognate substrates at the active site of class II lysyl-tRNA synthetase

Within the two unrelated aminoacyl-tRNA synthetase classes, lysyl-tRNA synthetase (LysRS) is the only example known to exist in both classes. To probe the role of the amino acids responsible for L-lysine binding in the active site of the class II LysRS (LysRS2), we studied the lysS-encoded Escherichia coli protein. On the basis of the structure of L-lysine complexed with E. coli LysRS2 (lysS), residues implicated in amino acid recognition and discrimination were systematically replaced. Steady-state kinetic parameters for these variants showed reductions in the catalytic efficiency (k(cat)/K(M)) of 1-3 orders of magnitude, allowing the assignment of specific roles for key residues in the active site of LysRS2. To further investigate the role of each residue in discrimination against noncognate amino acids, steady-state kinetic parameters were determined for the nonprotein amino acid S-(2-aminoethyl)-L-cysteine, a potent inhibitor of LysRS2. While a number of variants showed reductions of several hundred-fold in efficiency of S-(2-aminoethyl)-L-cysteine utilization, this was uniformly accompanied by similar reductions in the efficiency of lysine utilization. Thus, manipulation of the amino acid binding site only allowed up to a 4-fold improvement in S-(2-aminoethyl)-L-cysteine discrimination. This is in contrast to the highly effective discrimination against S-(2-aminoethyl)-L-cysteine by class I LysRS and correlates with the fundamentally different roles of conserved aromatic residues in the two LysRS active sites. This now provides a mechanistic basis for the proposal that differences in amino acid discrimination have been pivotal in the evolution of two unrelated LysRSs.     

Transfer RNA recognition by class I lysyl-tRNA synthetase from the Lyme disease pathogen Borrelia burgdorferi

Borrelia burgdorferi and other spirochetes contain a class I lysyl-tRNA synthetase (LysRS), in contrast to most eubacteria that have a canonical class II LysRS. We analyzed tRNA(Lys) recognition by B. burgdorferi LysRS, using two complementary approaches. First, the nucleotides of B. burgdorferi tRNA(Lys) in contact with B. burgdorferi LysRS were determined by enzymatic footprinting experiments. Second, the kinetic parameters for a series of variants of the B. burgdorferi tRNA(Lys) were then determined during aminoacylation by B. burgdorferi LysRS. The identity elements were found to be mostly located in the anticodon and in the acceptor stem. Transplantation of the identified identity elements into the Escherichia coli tRNA(Asp) scaffold endowed lysylation activity on the resulting chimera, indicating that a functional B. burgdorferi lysine tRNA identity set had been determined.     

The N-terminal domain of mammalian Lysyl-tRNA synthetase is a functional tRNA-binding domain.

Lysyl-tRNA synthetase from higher eukaryotes possesses a lysine-rich N-terminal polypeptide extension appended to a classical prokaryotic-like LysRS domain. Band shift analysis showed that this extra domain provides LysRS with nonspecific tRNA binding properties. A N-terminally truncated derivative of LysRS, LysRS-DeltaN, displayed a 100-fold lower apparent affinity for tRNA(3)Lys and a 3-fold increase in K(m) for tRNA(3)Lys in the aminoacylation reaction, as compared with the native enzyme. The isolated N-domain of LysRS also displayed weak affinity for tRNA, suggesting that the catalytic and N-domains of LysRS act synergistically to provide a high affinity binding site for tRNA. A more detailed analysis revealed that LysRS binds and specifically aminoacylates an RNA minihelix mimicking the amino acid acceptor stem-loop structure of tRNA(3)Lys, whereas LysRS-DeltaN did not. As a consequence, merging an additional RNA-binding domain into a bacterial-like LysRS increases the catalytic efficiency of the enzyme, especially at the low concentration of deacylated tRNA prevailing in vivo. Our results provide new insights into tRNA(Lys) channeling in eukaryotic cells and shed new light on the possible requirement of native LysRS for triggering tRNA(3)Lys packaging into human immunodeficiency virus, type 1 viral particles.     

Hearing impairment-associated KARS mutations lead to defects in aminoacylation of both cytoplasmic and mitochondrial tRNALys

Aminoacyl-tRNA synthetases(aaRSs) are ubiquitously expressed, essential enzymes, synthesizing aminoacyl-tRNAs for protein synthesis. Functional defects of aaRSs frequently cause various human disorders. Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases(LysRSs). Previously, two mutations(c.1129 GA and c.517 TC) were identified that led to hearing impairment; however, the underlying biochemical mechanism is unclear. In the present study, we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol, but affect the cytosolic LysRS level, its tertiary structure, and cytosolic tRNA aminoacylation in vitro. As for mitochondrial translation, the two mutations have little effect on the steady-state level, mitochondrial targeting, and tRNA binding affinity of mitochondrial LysRS. However, they exhibit striking differences in charging mitochondrial tRNA~(Lys), with the c.517TC mutant being completely deficient in vitro and in vivo. We constructed two yeast genetic models, which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels. Overall, our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129GA and c.517TC mutations and provided yeast genetic bases to investigate other KARS mutations in the future.     

Identity elements for specific aminoacylation of a tRNA by mammalian lysyl-tRNA synthetase bearing a nonspecific tRNA-interacting factor

Mammalian lysyl-tRNA synthetase (LysRS) has an N-terminal polypeptide chain extension appended to a prokaryotic-like synthetase domain. This extension, termed a tRNA-interacting factor (tIF), possesses a RNA-binding motif [KxxxK(K/R)xxK] that binds nonspecifically the acceptor TPsiC stem-loop domain of tRNA and provides a potent tRNA binding capacity to this enzyme. Consequently, native LysRS aminoacylates a RNA minihelix mimicking the amino acid acceptor stem-loop domain of tRNA(3)(Lys). Here, examination of minihelix recognition showed that mammalian LysRS aminoacylates RNA minihelices without specificity of sequence, revealing that none of the nucleotides from the acceptor TPsiC stem-loop domain are essential determinants of tRNA(Lys) acceptor identity. To test whether the tIF domain reduces the specificity of the synthetase with regard to complete tRNA molecules, aminoacylation of wild-type and mutant noncognate tRNAs by wild-type or N-terminally truncated LysRS was examined. The presence of the UUU anticodon of tRNA(Lys) appeared to be necessary and sufficient to transform yeast tRNA(Asp) or tRNA(i)(Met) into potent lysine acceptor tRNAs. Thus, nonspecific RNA-protein interactions between the acceptor stem of tRNA and the tIF domain do not relax the tRNA specificity of mammalian LysRS. The possibility that interaction of the full-length cognate tRNA with the synthetase is required to induce the catalytic center of the enzyme into a productive conformation is discussed.     

Anticodon recognition and discrimination by the alpha-helix cage domain of class I lysyl-tRNA synthetase

Aminoacyl-tRNA synthetases are normally found in one of two mutually exclusive structural classes, the only known exception being lysyl-tRNA synthetase which exists in both classes I (LysRS1) and II (LysRS2). Differences in tRNA acceptor stem recognition between LysRS1 and LysRS2 do not drastically impact cellular aminoacylation levels, focusing attention on the mechanism of tRNA anticodon recognition by LysRS1. On the basis of structure-based sequence alignments, seven tRNALys anticodon variants and seven LysRS1 anticodon binding site variants were selected for analysis of the Pyrococcus horikoshii LysRS1-tRNALys docking model. LysRS1 specifically recognized the bases at positions 35 and 36, but not that at position 34. Aromatic residues form stacking interactions with U34 and U35, and aminoacylation kinetics also identified direct interactions between Arg502 and both U35 and U36. Tyr491 was also found to interact with U36, and the Y491E variant exhibited significant improvement compared to the wild type in aminoacylation of a tRNALysUUG mutant. Refinement of the LysRS1-tRNALys docking model based upon these data suggested that anticodon recognition by LysRS1 relies on considerably fewer interactions than that by LysRS2, providing a structural basis for the more significant role of the anticodon in tRNA recognition by the class II enzyme. To date, only glutamyl-tRNA synthetase (GluRS) has been found to contain an alpha-helix cage anticodon binding domain homologous to that of LysRS1, and these data now suggest that specificity for the anticodon of tRNALys could have been acquired through relatively few changes to the corresponding domain of an ancestral GluRS enzyme.     

Functional association between three archaeal aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching amino acids to their cognate tRNAs during protein synthesis. In eukaryotes aaRSs are commonly found in multi-enzyme complexes, although the role of these complexes is still not completely clear. Associations between aaRSs have also been reported in archaea, including a complex between prolyl-(ProRS) and leucyl-tRNA synthetases (LeuRS) in Methanothermobacter thermautotrophicus that enhances tRNA(Pro) aminoacylation. Yeast two-hybrid screens suggested that lysyl-tRNA synthetase (LysRS) also associates with LeuRS in M. thermautotrophicus. Co-purification experiments confirmed that LeuRS, LysRS, and ProRS associate in cell-free extracts. LeuRS bound LysRS and ProRS with a comparable K(D) of about 0.3-0.9 microm, further supporting the formation of a stable multi-synthetase complex. The steady-state kinetics of aminoacylation by LysRS indicated that LeuRS specifically reduced the Km for tRNA(Lys) over 3-fold, with no additional change seen upon the addition of ProRS. No significant changes in aminoacylation by LeuRS or ProRS were observed upon the addition of LysRS. These findings, together with earlier data, indicate the existence of a functional complex of three aminoacyl-tRNA synthetases in archaea in which LeuRS improves the catalytic efficiency of tRNA aminoacylation by both LysRS and ProRS.     

NMR Structure of the C-terminal domain of a tyrosyl-tRNA synthetase that functions in group I intron splicing

The mitochondrial tyrosyl-tRNA synthetases (mt TyrRSs) of Pezizomycotina fungi are bifunctional proteins that aminoacylate mitochondrial tRNA(Tyr) and are structure-stabilizing splicing cofactors for group I introns. Studies with the Neurospora crassa synthetase (CYT-18 protein) showed that splicing activity is dependent upon Pezizomycotina-specific structural adaptations that form a distinct group I intron-binding site in the N-terminal catalytic domain. Although CYT-18's C-terminal domain also binds group I introns, it has been intractable to X-ray crystallography in the full-length protein. Here, we determined an NMR structure of the isolated C-terminal domain of the Aspergillus nidulans mt TyrRS, which is closely related to but smaller than CYT-18's. The structure shows an S4 fold like that of bacterial TyrRSs, but with novel features, including three Pezizomycontia-specific insertions. (15)N-(1)H two-dimensional NMR showed that C-terminal domains of the full-length A. nidulans and Geobacillus stearothermophilus synthetases do not tumble independently in solution, suggesting restricted orientations. Modeling onto a CYT-18/group I intron cocrystal structure indicates that the C-terminal domains of both subunits of the homodimeric protein bind different ends of the intron RNA, with one C-terminal domain having to undergo a large shift on its flexible linker to bind tRNA(Tyr) or the intron RNA on either side of the catalytic domain. The modeling suggests that the C-terminal domain acts together with the N-terminal domain to clamp parts of the intron's catalytic core, that at least one C-terminal domain insertion functions in group I intron binding, and that some C-terminal domain regions bind both tRNA(Tyr) and group I intron RNAs.     

Discrimination of cognate and noncognate substrates at the active site of class I lysyl-tRNA synthetase

The aminoacyl-tRNA synthetases are divided into two unrelated structural classes, with lysyl-tRNA synthetase (LysRS) being the only enzyme represented in both classes. On the basis of the structure of l-lysine complexed with Pyrococcus horikoshii class I LysRS (LysRS1) and homology to glutamyl-tRNA synthetase (GluRS), residues implicated in amino acid recognition and noncognate substrate discrimination were systematically replaced in Borrelia burgdorferi LysRS1. The catalytic efficiency of steady-state aminoacylation (k(cat)/K(M)) with lysine by LysRS1 variants fell by 1-4 orders of magnitude compared to that of the wild type. Disruption of putative hydrogen bonding interactions through replacement of G29, T31, and Y269 caused up to 1500-fold reductions in k(cat)/K(M), similar to changes previously observed for comparable variants of class II LysRS (LysRS2). Replacements of W220 and H242, both of which are implicated in hydrophobic interactions with the side chain of lysine, resulted in more dramatic changes with up to 40000-fold reductions in k(cat)/K(M) observed. This indicates that the more compact LysRS1 active site employs both electrostatic and hydrophobic interactions during lysine discrimination, explaining the ability of LysRS1 to discriminate against noncognate substrates accepted by LysRS2. Several of the LysRS1 variants were found to be more specific than the wild type with respect to noncognate amino acid recognition but less efficient in cognate aminoacylation. This indicates that LysRS1 compromises between efficient catalysis and substrate discrimination, in contrast to LysRS2 which is considerably more effective in catalysis but is less specific than its class I counterpart.     

The T box regulatory element controlling expression of the class I lysyl-tRNA synthetase of <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain 14579 is functional and can be partially induced by reduced charging of asparaginyl-tRNA<Superscript>Asn</Superscript>

Background  

Lysyl-tRNA synthetase (LysRS) is unique within the aminoacyl-tRNA synthetase family in that both class I (LysRS1) and class II (LysRS2) enzymes exist. LysRS1 enzymes are found in Archaebacteria and some eubacteria while all other organisms have LysRS2 enzymes. All sequenced strains of Bacillus cereus (except AH820) and Bacillus thuringiensis however encode both a class I and a class II LysRS. The lysK gene (encoding LysRS1) of B. cereus strain 14579 has an associated T box element, the first reported instance of potential T box control of LysRS expression.     

Function independence of microhelix aminoacylation from anticodon binding in a class I tRNA synthetase.

The monomeric form of the class I Escherichia coli methionine tRNA synthetase has a distinct carboxyl-terminal domain with a segment that interacts with the anticodon of methionine tRNA. This interaction is a major determinant of the specificity and efficiency of aminoacylation. The end of this carboxyl-terminal domain interacts with the amino-terminal Rossman fold that forms the site for amino acid activation. Thus, the carboxyl-terminal end may have evolved in part to integrate anticodon recognition with amino acid activation. We show here that internal deletions that disrupt the anticodon interaction have no effect on the kinetic parameters for amino acid activation. Moreover, an internally deleted enzyme can aminoacylate an RNA microhelix, which is based on the acceptor stem of methionine tRNA, with the same efficiency as the native protein. These results suggest that, in this enzyme, amino acid activation and acceptor helix aminoacylation are functionally integrated and are independent of the anticodon-binding site.