Dual mode recognition of two isoacceptor tRNAs by mammalian mitochondrial seryl-tRNA synthetase

Animal mitochondrial translation systems contain two serine tRNAs, corresponding to the codons AGY (Y = U and C) and UCN (N = U, C, A, and G), each possessing an unusual secondary structure; tRNA(GCU)(Ser) (for AGY) lacks the entire D arm, whereas tRNA(UGA)(Ser) (for UCN) has an unusual cloverleaf configuration. We previously demonstrated that a single bovine mitochondrial seryl-tRNA synthetase (mt SerRS) recognizes these topologically distinct isoacceptors having no common sequence or structure. Recombinant mt SerRS clearly footprinted at the TPsiC loop of each isoacceptor, and kinetic studies revealed that mt SerRS specifically recognized the TPsiC loop sequence in each isoacceptor. However, in the case of tRNA(UGA)(Ser), TPsiC loop-D loop interaction was further required for recognition, suggesting that mt SerRS recognizes the two substrates by distinct mechanisms. mt SerRS could slightly but significantly misacylate mitochondrial tRNA(Gln), which has the same TPsiC loop sequence as tRNA(UGA)(Ser), implying that the fidelity of mitochondrial translation is maintained by kinetic discrimination of tRNAs in the network of aminoacyl-tRNA synthetases.     

The contacts of yeast tRNA(Ser) with seryl-tRNA synthetase studied by footprinting experiments

Yeast tRNA(Ser) is a member of the class II tRNAs, whose characteristic is the presence of an extended variable loop. This additional structural feature raises questions about the recognition of these class II tRNAs by their cognate synthetase and the possibility of the involvement of the extra arm in the recognition process. A footprinting study of yeast tRNA(Ser) complexed with its cognate synthetase, yeast seryl-tRNA synthetase (an alpha 2 dimer), was undertaken. Chemical (ethylnitrosourea) and enzymatic (nucleases S1 and V1) probes were used in the experiments. A map of the contact points between the tRNA and the synthetase was established and results were analyzed with respect to a three-dimensional model of yeast tRNA(Ser). Regions in close vicinity with the synthetase are clustered on one face of tRNA. The extra arm, which is strongly protected from chemical modifications, appears as an essential part of the contact area. The anticodon triplet and a large part of the anticodon arm are, in contrast, still accessible to the probes when the complex is formed. These results are discussed in the context of the recognition of tRNAs in the aminoacylation reaction.     

Variant minihelix RNAs reveal sequence-specific recognition of the helical tRNA(Ser) acceptor stem by E.coli seryl-tRNA synthetase.

Aminoacylation rate determinations for a series of variant RNA minihelix substrates revealed that Escherichia coli seryl-tRNA synthetase (SerRS) recognizes the 1--72 through 5--68 base pairs of the E.coli tRNA(Ser) acceptor stem with the major recognition elements clustered between positions 2--71 and 4--69. The rank order of effects of canonical base pair substitutions at each position on kcat/Km was used to assess the involvement of major groove functional groups in recognition. Conclusions based on the biochemical data are largely consistent with the interactions revealed by the refined structure of the homologous Thermus thermophilus tRNA(Ser)-SerRS complex that Cusack and colleagues report in the accompanying paper. Disruption of an end-on hydrophobic interaction between the major groove C5(H) of pyrimidine 69 and an aromatic side chain of SerRS is shown to significantly decrease kcat/Km of a minihelix substrate. This type of interaction provides a means by which proteins can recognize the binary information of 'degenerate' sequences, such as the purine-pyrimidine base pairs of tRNA(Ser). The 3--70 base pair is shown to contribute to recognition by SerRS even though it is not contacted specifically by the protein. The latter effect derives from the organization of the specific contacts that SerRS makes with the neighboring 2--71 and 4--69 acceptor stem base pairs.     

Recognition of tRNAs by Methionyl-tRNA transformylase from mammalian mitochondria

Protein synthesis involves two methionine-isoaccepting tRNAs, an initiator and an elongator. In eubacteria, mitochondria, and chloroplasts, the addition of a formyl group gives its full functional identity to initiator Met-tRNA(Met). In Escherichia coli, it has been shown that the specific action of methionyl-tRNA transformylase on Met-tRNA(f)(Met) mainly involves a set of nucleotides in the acceptor stem, particularly a C(1)A(72) mismatch. In animal mitochondria, only one tRNA(Met) species has yet been described. It is admitted that this species can engage itself either in initiation or elongation of translation, depending on the presence or absence of a formyl group. In the present study, we searched for the identity elements of tRNA(Met) that govern its formylation by bovine mitochondrial transformylase. The main conclusion is that the mitochondrial formylase preferentially recognizes the methionyl moiety of its tRNA substrate. Moreover, the relatively small importance of the tRNA acceptor stem in the recognition process accounts for the protection against formylation of the mitochondrial tRNAs that share with tRNA(Met) an A(1)U(72) motif.     

An orthogonal seryl-tRNA synthetase/tRNA pair for noncanonical amino acid mutagenesis in Escherichia coli

We report the development of the orthogonal amber-suppressor pair Archaeoglobus fulgidus seryl-tRNA (Af-tRNA^Ser)/Methanosarcina mazei seryl-tRNA synthetase (MmSerRS) in Escherichia coli. Furthermore, the crystal structure of MmSerRS was solved at 1.45 Å resolution, which should enable structure-guided engineering of its active site to genetically encode small, polar noncanonical amino acids (ncAAs).     

Dual targeting of organellar seryl-tRNA synthetase to maize mitochondria and chloroplasts

Aminoacyl-tRNA synthetases (AARSs) play a critical role in translation and are thus required in three plant protein-synthesizing compartments: cytosol, mitochondria and plastids. A systematic study had previously shown extensive sharing of organellar AARSs from Arabidopsis thaliana, mostly between mitochondria and chloroplasts. However, distribution of AARSs from monocot species, such as maize, has never been experimentally investigated. Here we demonstrate dual targeting of maize seryl-tRNA synthetase, SerZMo, into both mitochondria and chloroplasts using combination of complementary methods, including in vitro import assay, transient expression analysis of green fluorescent protein (GFP) fusions and immunodetection. We also show that SerZMo dual localization is established by the virtue of an ambiguous targeting peptide. Full-length SerZMo protein fused to GFP is targeted to chloroplast stromules, indicating that SerZMo protein performs its function in plastid stroma. The deletion mutant lacking N-terminal region of the ambiguous SerZMo targeting peptide was neither targeted into mitochondria nor chloroplasts, indicating the importance of this region in both mitochondrial and chloroplastic import.     

Structure of the unusual seryl-tRNA synthetase reveals a distinct zinc-dependent mode of substrate recognition

Methanogenic archaea possess unusual seryl-tRNA synthetase (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. The two types of SerRSs show only minimal sequence similarity, primarily within class II conserved motifs 1, 2 and 3. Here, we report a 2.5 A resolution crystal structure of the atypical methanogenic Methanosarcina barkeri SerRS and its complexes with ATP, serine and the nonhydrolysable seryl-adenylate analogue 5'-O-(N-serylsulfamoyl)adenosine. The structures reveal two idiosyncratic features of methanogenic SerRSs: a novel N-terminal tRNA-binding domain and an active site zinc ion. The tetra-coordinated Zn2+ ion is bound to three conserved protein ligands (Cys306, Glu355 and Cys461) and binds the amino group of the serine substrate. The absolute requirement of the metal ion for enzymatic activity was confirmed by mutational analysis of the direct zinc ion ligands. This zinc-dependent serine recognition mechanism differs fundamentally from the one employed by the bacterial-type SerRSs. Consequently, SerRS represents the only known aminoacyl-tRNA synthetase system that evolved two distinct mechanisms for the recognition of the same amino-acid substrate.     

Influence of various factors on the recognition specificity of tRNAs by yeast valyl-tRNA synthetase.

Using filtration through nitrocellulose membranes we found that complexes between yeast valyl-tRNA synthetase can easily be detected at low pH and ionic strength with the cognate tRNAVal, but also with several non-cognate tRNAs (tRNAPhe, tRNATyr, tRNAMet and tRNAAsp). We show here that the amino acid linked to the tRNA has no detectable effect on these interactions. The influence of various factors on the discrimination by the enzyme between the cognate and the non-cognate tRNAs has been studied. An increase in pH or ionic strength leads to a decrease in the same ratio of the affinity constants between the enzyme and the cognate as well as the noncognate tRNA. The addition of organic solvents has little effect on these constant either in the cognate or in the non-cognate systems; the addition of substrates of the aminoacylation reaction has not effect on the ratio between the constants. This similar behaviour suggests that at least part of the specific of non-specific interactions must be identical. On the contrary, magnesium between 1 mM and 50 mM increases the specificity of recognition, showing the importance of slight conformational changes in the tRNA molecule to the specificity of interaction.     

A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution.

Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.     

Peroxin Pex21p interacts with the C-terminal noncatalytic domain of yeast seryl-tRNA synthetase and forms a specific ternary complex with tRNA(Ser)

The seryl-tRNA synthetase from Saccharomyces cerevisiae interacts with the peroxisome biogenesis-related factor Pex21p. Several deletion mutants of seryl-tRNA synthetase were constructed and inspected for their ability to interact with Pex21p in a yeast two-hybrid assay, allowing mapping of the synthetase domain required for complex assembly. Deletion of the 13 C-terminal amino acids abolished Pex21p binding to seryl-tRNA synthetase. The catalytic parameters of purified truncated seryl-tRNA synthetase, determined in the serylation reaction, were found to be almost identical to those of the native enzyme. In vivo loss of interaction with Pex21p was confirmed in vitro by coaffinity purification. These data indicate that the C-terminally appended domain of yeast seryl-tRNA synthetase does not participate in substrate binding, but instead is required for association with Pex21p. We further determined that Pex21p does not directly bind tRNA, and nor does it possess a tRNA-binding motif, but it instead participates in the formation of a specific ternary complex with seryl-tRNA synthetase and tRNA(Ser), strengthening the interaction of seryl-tRNA synthetase with its cognate tRNA(Ser).     

Purification and properties of phosphatidylglycerophosphate synthetase from mammalian liver mitochondria

The enzyme which catalyzes the synthesis of phosphatidylgly cerophosphate from an-glycerol-3-phosphated and cytidine diphosphate diacylglycerol was released from rat or pig liver mitochondrial membranes by extraction with Triton X-100 or Nonidet P-40. The detergent-extracted enzyme, like the activity of intact mitochondria, did not require added cations or lipids. The Triton extracts were fractionated by column chromatography on Bio-Gel A-1.5. The fractions obtained from the columns exhibited little activity in the standard assay system unless divalent cations were included. Additional stimulation (about twofold) was observed in the presence of added phospholipids. The cation requirement of the purified enzyme was relatively nonspecific with Mg2+, Ba2+, or Ca2+ providing maximal activity in the 10mM range. Either Mn2+ or Co2+ were stimulatory at somewhat lower concentrations but higher concentrations were inhibitory. Other cations such as Cd2+, Zn2+,Hg2+, or Cu2+ were ineffective as cofactors, and in the presence of Mg2+ inhibited the reaction at concentrations greater than 0.5 mM. The phospholipik stimulation was obtained specifically with phosphatidylethanolamines from natural or synthetic sources. Other diacylglycerophosphatides or lysophosphatides including lysophosphatidylethanolamine were ineffective.     

Enhancement of binding of N-hydroxy-Trp-P-2 to DNA by seryl-tRNA synthetase

Covalent binding to DNA of a mutagenic metabolite of Trp-P-2, N-hydroxy-Trp-P-2, was examined in the presence of seryl-tRNA synthetase. Both ATP and L-serine were essential requirements for this binding. In the absence of seryl-tRNA synthetase, the binding was reduced to about 14% of the complete system. These results indicate that seryl-tRNA synthetase which is widely distributed in tissues of experimental animals might act as the activating enzyme of N-hydroxy-Trp-P-2.     

The aminoacylation of structurally variant phenylalanine tRNAs from mitochondria and various nonmitochondrial sources by bovine mitochondrial phenylalanyl-tRNA synthetase

Bovine mitochondrial (mt) phenylalanine tRNA (tRNAPhe) was purified on a large scale using a new hybridization assay method developed by the authors. Although its melting profile suggested a loose higher order structure, presumably influenced by the apparent loss of D loop-T loop interaction necessary for forming a rigid L-shaped tertiary structure, its aminoacylation capacity catalyzed by mt phenylalanyl-tRNA synthetase (PheRS) was nearly equal to that of Escherichia coli tRNAPhe. Misaminoacylation was not observed for the mt tRNAPhe-mt PheRS system. Comparing the aminoacylation efficiencies of several combinations of tRNAPheS and PheRSs from various sources, including bovine mitochondria, bovine and yeast cytosols, E. coli, Thermus thermophilus, and Sulfolobus acidocaldarius, it was clarified that mt PheRS was able to aminoacylate all the above mentioned tRNAPhe species, albeit with varying degrees of efficiency. This broad charging spectrum suggests that mt PheRS possesses a relatively simple recognition mechanism toward its substrate, tRNAPhe.