Construction of a FRS1-FRS2 operon encoding the structural genes for the alpha and beta subunits of yeast phenylalanyl-tRNA synthetase and its use in deletion analysis.

FRS1 and FRS2, the structural genes encoding the large (alpha) and small (beta) subunits of yeast phenylalanyl-tRNA synthetase (PheRS) were placed under the control of the lacZ promoter by creating an artificial operon. The FRS2 gene was fused next to the promoter, followed by a 14 base pair intergenic sequence containing a translation reinitiation site in front of the FRS1 coding sequences. The engineered PheRS has 16 N-terminal amino acids from beta-galactosidase fused to the beta subunit. However, the purified protein shows a Km value for tRNA(Phe) that is indistinguishable from that of the the native enzyme. The product of the FRS2-FRS1 operon is not able to complement thermosensitive E. coli PheRS, indicating the lack of heterologous aminoacylation in vivo. We made a deletion in the FRS2 gene that removed about 150 amino terminal residues of the beta subunit. The truncated protein showed intact ATP-PPi exchange, whereas tRNA aminoacylation was lost. This result is similar to that of limited proteolysis performed on the native enzyme that yielded a tetrameric alpha 2 beta'2 structure, able to form aminoacyladenylate but unable to bind tRNA(Phe). A deletion of 50 amino acids from the carboxyl terminus of the beta chain resulted in the loss of both enzyme activities; this suggests the participation of the C-terminal end of the beta subunit in the active site or in subunit assembly to yield a tetrameric functional enzyme.     

Leucyl-tRNA synthetase consisting of two subunits from hyperthermophilic bacteria Aquifex aeolicus

In a hyperthermophilic bacterium, Aquifex aeolicus, leucyl-tRNA synthetase (LeuRS) consists of two non-identical polypeptide subunits (alpha and beta), different from the canonical LeuRS, which has a single polypeptide chain. By PCR, using genome DNA of A. aeolicus as a template, genes encoding the alpha and beta subunits were amplified and cloned in Escherichia coli. The alpha subunit could not be expressed stably in vivo, whereas the beta subunit was overproduced and purified by a simple procedure. The beta subunit was inactive in catalysis but was able to bind tRNA(Leu). Interestingly, the heterodimer alphabeta-LeuRS could be overproduced in E. coli cells containing both genes and was purified to 95% homogeneity as a hybrid dimer. The kinetics of A. aeolicus LeuRS in pre-steady and steady states and cross-recognition of LeuRS and tRNA(Leu) from A. aeolicus and E. coli were studied. Magnesium concentration, pH value, and temperature aminoacylation optima were determined to be 12 mm, 7.8, and 70 degrees C, respectively. Under optimal conditions, A. aeolicus alphabeta-LeuRS is stable up to 65 degrees C.     

Phenylalanyl-tRNA synthetase contains a dispensable RNA-binding domain that contributes to the editing of noncognate aminoacyl-tRNA

Phenylalanyl-tRNA synthetase (PheRS) is a multidomain (alphabeta)2 heterotetrameric protein responsible for synthesizing Phe-tRNA(Phe) during protein synthesis. Previous studies showed that the alpha subunit forms the catalytic core of the enzyme, while the beta subunit contains a number of autonomous structural modules with a wide range of functions including tRNA anticodon binding and editing of the misaminoacylated species Tyr-tRNA(Phe). The B2 domain of the beta subunit is a structural homologue of the EMAPII/OB fold, which has been shown in other systems to contribute to tRNA binding. Structural studies of PheRS indicated that the B2 domain is distant from bound tRNA(Phe), leaving the role of this module in question. On the basis of homology modeling with other EMAPII domain-containing proteins, the 110 amino acid B2 domain was deleted to produce PheRS deltaB2. Full-length PheRS and PheRS deltaB2 showed comparable kinetics for in vitro aminoacylation, and both enzymes complemented a defect in phenylalanylation in vivo. PheRS deltaB2 showed a 2-fold drop compared to full-length PheRS in the catalytic efficiency (kcat/KM) of Tyr-tRNA(Phe) hydrolysis, suggesting a role for the B2 domain in post-transfer editing. A comparison of tRNA binding by full-length PheRS and PheRS deltaB2 indicated that the B2 domain acts as a secondary tRNA-binding site that could contribute to editing by promoting the translocation of mischarged tRNA to the editing site of PheRS. This proposed role for the B2 domain of PheRS is consistent with previous studies, suggesting that the highly conserved EMAPII fold is able to modulate the affinity of tRNA for its primary binding site.     

Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNAPhe

Periodate-oxidized tRNA(Phe) (tRNA(oxPhe)) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the alpha 2 beta 2 enzyme with tRNA(oxPhe) results in the loss of tRNAPhe aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[14C]tRNA(oxPhe) covalent complex indicates that the large (alpha, Mr 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA(oxPhe). The [14C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Ile-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the alpha subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].     

Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations

Neither the tertiary structure nor the location of active sites are known for phenylalanyl-tRNA synthetase (PheRS; alpha 2 beta 2 structure), a member of class II aminoacyl-tRNA synthetases. In an attempt to detect the phenylalanine (Phe) binding site, two Escherichia coli PheRS mutant strains (pheS), which were resistant to p-fluorophenylalanine (p-F-Phe) were analysed genetically. The pheS mutations were found to cause Ala294 to Ser294 exchanges in the alpha subunits from both independent strains. This alteration (S294) resided in the well-conserved C-terminal part of the alpha subunit, precisely within motif 3, a typical class II tRNA synthetase sequence. We thus propose that motif 3 participates in the formation of the Phe binding site of PheRS. Mutation S294 was also the key for proposing a mechanism by which the substrate analogue p-F-Phe is excluded from the enzymatic reaction; this may be achieved by steric interactions between the para-position of the aromatic ring and the amino acid residue at position 294. The Phe binding site model was then tested by replacing the alanine at position 294 as well as the two flanking phenylalanines (positions 293 and 295) by a number of selected other amino acids. In vivo and in vitro results demonstrated that Phe293 and Phe295 are not directly involved in substrate binding, but replacements of those residues affected PheRS stability. However, exchanges at position 294 altered the binding of Phe, and certain mutants showed pronounced changes in specificity towards Phe analogues. Of particular interest was the Gly294 PheRS in which presumably an enlarged cavity for the para position of the aromatic ring allowed an increased aminoacylation of tRNA with p-F-Phe. Moreover, the larger para-chloro and para-bromo derivatives of Phe could interact with this enzyme in vitro and became highly toxic in vivo. The possible exploitation of the Gly294 mutant PheRS for the incorporation of non-proteinogenic amino acids into proteins is discussed.     

High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5 kDa, and named alphabeta-LeuRS. The gene encoding alpha subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of alpha subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNA(Arg)(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding beta subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce alphabeta-LeuRS with a His6 tag at the N-terminus of alpha subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7 mg purified alphabeta-LeuRS was obtained from 250 ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.     

Impaired affinity for phenylalanine in Escherichia coli phenylalanyl-tRNA synthetase mutant caused by Gly-to-Asp exchange in motif 2 of class II tRNA synthetases

Phenylalanyl-tRNA synthetase (PheRS; alpha 2 beta 2 subunit structure) is a member of class II of tRNA synthetases. We report here the genetic analysis of an Escherichia coli mutant strain which is auxotrophic for phenylalanine because it has a PheRS with a decreased affinity for phenylalanine. The mutant pheS gene encoding the PheRS alpha subunit was cloned and sequenced, and the deviation from the wild-type gene was found to result in a Gly191-to-Asp191 exchange. This alteration is located within motif 2, one of 3 conserved sequence motifs characteristic for class II aminoacyl-tRNA synthetases. Motif 2 may thus participate in the formation of the phenylalanine binding site in PheRS.     

Role of Low-Molecular-Weight Substrates in Functional Binding of the tRNAPhe Acceptor End by Phenylalanyl-tRNA Synthetase

The functional roles of phenylalanine and ATP in productive binding of the tRNA(Phe) acceptor end have been studied by photoaffinity labeling (cross-linking) of T. thermophilus phenylalanyl-tRNA synthetase (PheRS) with tRNA(Phe) analogs containing the s(4)U residue in different positions of the 3'-terminal single-stranded sequence. Human and E. coli tRNA(Phe)s used as basic structures differ by efficiency of the binding and aminoacylation with the enzyme under study. Destabilization of the complex with human tRNA(Phe) caused by replacement of three recognition elements decreases selectivity of labeling of the alpha- and beta-subunits responsible for the binding of adjacent nucleotides of the CCA-end. Phenylalanine affects the positioning of the base and ribose moieties of the 76th nucleotide, and the recorded effects do not depend on structural differences between bacterial and eukaryotic tRNA(Phe)s. Both in the absence and presence of phenylalanine, ATP more effectively inhibits the PheRS labeling with the s(4)U76-substituted analog of human tRNA(Phe) (tRNA(Phe)-s(4)U76) than with E. coli tRNA(Phe)-s(4)U76: in the first case the labeling of the alpha-subunits is inhibited more effectively; the labeling of the beta-subunits is inhibited in the first case and increased in the second case. The findings analyzed with respect to available structural data on the enzyme complexes with individual substrates suggest that the binding of phenylalanine induces a local rearrangement in the active site and directly controls positioning of the tRNA(Phe) 3'-terminal nucleotide. The effect of ATP on the acceptor end positioning is caused by global structural changes in the complex, which modulate the conformation of the acceptor arm. The rearrangement of the acceptor end induced by small substrates results in reorientation of the 3'-OH-group of the terminal ribose from the catalytic subunit onto the noncatalytic one, and this may explain the unusual stereospecificity of aminoacylation in this system.     

Expression,purification, and characterization of human tyrosyl-tRNA synthetase

Human tyrosyl-tRNA synthetase is a homodimeric enzyme and each subunit is near 58 KD. It catalyzes the aminoacylation of tRNA(Tyr) by L-tyrosine. The His(6)-tagged human TyrS gene was obtained by RT-PCR from total RNA of human lung giant-cell cancer strain 95 D. It was confirmed by sequencing and cloned into the expression vector pET-24 a (+) to yield pET-24 a (+)-HTyrRS, which was transfected into Escherichia coli BL21-CodonPlus-RIL. The induced-expression level of His(6)-tagged human TyrRS was about 24% of total cell proteins under IPTG inducing. The recombinant protein was conveniently purified in a single step by metal (Ni(2+)) chelate affinity chromatography. About 22.3mg purified enzyme could be obtained from 1L cell culture. The k(cat) value of His(6)-tagged human TyrRS in the second step of tRNA(Tyr) aminoacylation was 1.49 s(-1). The K(m) values of tyrosine and tRNA(Tyr) were 0.3 and 0.9 microM. Six His residues at the C terminus of human TyrRS have little effect on the activities of the enzyme compared with other eukaryotic TyrRSs.     

tRNA discrimination by T. thermophilus phenylalanyl-tRNA synthetase at the binding step

The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs. The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis. The anticodon nucleotides involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis. Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding. Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme. The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step. A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex. The highest affinity of T. thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs.     

The crystal structure of the ternary complex of phenylalanyl-tRNA synthetase with tRNAPhe and a phenylalanyl-adenylate analogue reveals a conformational switch of the CCA end

The crystal structure of the ternary complex of (alphabeta)(2) heterotetrameric phenylalanyl-tRNA synthetase (PheRS) from Thermus thermophilus with cognate tRNA(Phe) and a nonhydrolyzable phenylalanyl-adenylate analogue (PheOH-AMP) has been determined at 3.1 A resolution. It reveals conformational changes in tRNA(Phe) induced by the PheOH-AMP binding. The single-stranded 3' end exhibits a hairpin conformation in contrast to the partial unwinding observed previously in the binary PheRS.tRNA(Phe) complex. The CCA end orientation is stabilized by extensive base-specific interactions of A76 and C75 with the protein and by intra-RNA interactions of A73 with adjacent nucleotides. The 4-amino group of the "bulged out" C75 is trapped by two negatively charged residues of the beta subunit (Glubeta31 and Aspbeta33), highly conserved in eubacterial PheRSs. The position of the A76 base is stabilized by interactions with Hisalpha212 of motif 2 (universally conserved in PheRSs) and class II-invariant Argalpha321 of motif 3. Important conformational changes induced by the binding of tRNA(Phe) and PheOH-AMP are observed in the catalytic domain: the motif 2 loop and a "helical" loop (residues 139-152 of the alpha subunit) undergo coordinated displacement; Metalpha148 of the helical loop adopts a conformation preventing the 2'-OH group of A76 from approaching the alpha-carbonyl carbon of PheOH-AMP. The unfavorable position of the terminal ribose stems from the absence of the alpha-carbonyl oxygen in the analogue. Our data suggest that the idiosyncratic feature of PheRS, which aminoacylates the 2'-OH group of the terminal ribose, is dictated by the system-specific topology of the CCA end-binding site.     

Prokaryotic and eukaryotic tetrameric phenylalanyl-tRNA synthetases display conservation of the binding mode of the tRNA(Phe) CCA end

The interaction of human phenylalanyl-tRNA synthetase, a eukaryotic prototype with an unknown three-dimensional structure, with the tRNA(Phe) acceptor end was studied by s(4)U-induced affinity cross-linking with human tRNA(Phe) derivatives site-specifically substituted at the single-stranded 3' end. Two different subunits of the enzyme bind two adjacent nucleotides of the tRNA(Phe) 3' end: nucleotide 76 is associated with the catalytic alpha subunit, while nucleotide 75 is in contact with the beta subunit. The binding mode is similar to that revealed previously in structural and affinity cross-linking studies of the prokaryotic Thermus thermophilus phenylalanyl-tRNA synthetase. Our results suggest that the distinctive features of tRNA(Phe) acceptor end binding are conserved for the eukaryotic and prokaryotic tetrameric phenylalanyl-tRNA synthetases despite their significant differences in the domain composition of the beta subunits. The data from affinity cross-linking experiments with human phenylalanyl-tRNA synthetase complexed with small ligands (ATP and/or phenylalanine or a stable synthetic analogue of phenylalanyl adenylate) reveal that the location of the tRNA(Phe) acceptor end varies with the presence and nature of other substrates. The lack of substrate activity of human tRNA(Phe) substituted with s(4)U at the 3'-terminal position suggests that base-specific interactions of the terminal adenosine are critically important for a productive interaction. The conformational rearrangement of the tRNA 3' end induced by the other substrates and dictated by base-specific contacts of the terminal nucleotide is an additional means of ensuring the phenylalanylation specificity in both prokaryotic and eukaryotic systems.     

Large-scale movement of functional domains facilitates aminoacylation by human mitochondrial phenylalanyl-tRNA synthetase

Structural studies suggest rearrangement of the RNA-binding and catalytic domains of human mitochondrial PheRS (mtPheRS) is required for aminoacylation. Crosslinking the catalytic and RNA-binding domains resulted in a “closed” form of mtPheRS that still catalyzed ATP-dependent Phe activation, but was no longer able to transfer Phe to tRNA and complete the aminoacylation reaction. SAXS experiments indicated the presence of both the closed and open forms of mtPheRS in solution. Together, these results indicate that conformational flexibility of the two functional modules in mtPheRS is essential for its phenylalanylation activity. This is consistent with the evolution of the aminoacyl-tRNA synthetases as modular enzymes consisting of separate domains that display independent activities.     

Enzymes assembled from Aquifex aeolicus and Escherichia coli leucyl-tRNA synthetases

Aquifex aeolicus alphabeta-LeuRS is the only known heterodimeric LeuRS, while Escherichia coli LeuRS is a canonical monomeric enzyme. By using the genes encoding A. aeolicus and E. coli LeuRS as PCR templates, the genes encoding the alpha and beta subunits from A. aeolicus alphabeta-LeuRS and the equivalent amino- and carboxy-terminal parts of E. coli LeuRS (identified as alpha' and beta') were amplified and recombined using suitable plasmids. These recombinant plasmids were transformed or cotransformed into E. coli to produce five monomeric and five heterodimeric LeuRS mutants. Seven of these were successfully overexpressed in vivo and purified, while three dimeric mutants with the beta' part of E. coli LeuRS were not successfully expressed. The seven purified mutants catalyzed amino acid activation, although several exhibited reduced aminoacylation properties. Removal of the last 36 residues of the alpha subunit of the A. aeolicus enzyme was determined to be deleterious for tRNA charging. Indeed, subunit exchange showed that the cross-species-specific recognition of A. aeolicus tRNA(Leu) occurs at the alpha subunit. None of the mixed E. coli-A. aeolicus enzymes were as thermostable as the native alphabeta-LeuRS. However, the fusion of the two alpha and beta peptides from A. aeolicus as a single chain analogous to canonical LeuRS resulted in a product more resistant to heat denaturation than the original enzyme.     

Promoting the formation of an active synthetase/tRNA complex by a nonspecific tRNA-binding domain

Previous studies showed that valyl-tRNA synthetase of Saccharomyces cerevisiae contains an N-terminal polypeptide extension of 97 residues, which is absent from its bacterial relatives, but is conserved in its mammalian homologues. We showed herein that this appended domain and its human counterpart are both nonspecific tRNA-binding domains (K(d) approximately 0.5 microm). Deletion of the appended domain from the yeast enzyme severely impaired its tRNA binding, aminoacylation, and complementation activities. This N-domain-deleted yeast valyl-tRNA synthetase mutant could be rescued by fusion of the equivalent domain from its human homologue. Moreover, fusion of the N-domain of the yeast enzyme or its human counterpart to Escherichia coli glutaminyl-tRNA synthetase enabled the otherwise "inactive" prokaryotic enzyme to function as a yeast enzyme in vivo. Different from the native yeast enzyme, which showed different affinities toward mixed tRNA populations, the fusion enzyme exhibited similar binding affinities for all yeast tRNAs. These results not only underscore the significance of nonspecific tRNA binding in aminoacylation, but also provide insights into the mechanism of the formation of aminoacyl-tRNAs.     

Expression and characterization of a human mitochondrial phenylalanyl-tRNA synthetase.

Human mitochondrial phenylalanyl-tRNA synthetase (mtPheRS) has been identified from the human EST database. Using consensus sequences derived from conserved regions of the alpha and beta-subunits from bacterial PheRS, two partially sequenced cDNA clones were identified. Unexpectedly, sequence analysis indicated that one of these clones was a truncated form of the other. Detailed analysis indicates that unlike the (alphabeta)2 structure of the prokaryotic and eukaryotic cytoplasmic forms of PheRS, the human mtPheRS consists of a single polypeptide chain. This protein has been cloned and expressed in Escherichia coli. Gel filtration and analytical velocity sedimentation centrifugation indicate that the human mtPheRS is active in a monomeric form. The N-terminal 314 amino acid residues appear to be analogous to the alpha-subunit of the prokaryotic PheRS, while the C-terminal 100 amino acid residues correspond to a region of the beta-subunit known to interact with the anticodon of tRNAPhe. Comparisons with the sequences of PheRS from yeast and Drosophila mitochondria indicate they are 42 % and 51 % identical with the human mtPheRS, respectively. Sequence analysis confirms the presence of motifs characteristic of class II aminoacyl-tRNA synthetases. KM and kcat values for ATP:PPi exchange and for the aminoacylation reaction carried out by human mtPheRS have been determined. Evolutionary origins of this small monomeric human mtPheRS are unknown, however, implications are that this enzyme is a result of the simplification of the more complex (alphabeta)2 bacterial PheRS in which specific functional regions were retained.     

Identification of the major tRNA(Phe) binding domain in the tetrameric structure of cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast

Native cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast is a tetramer of the alpha 2 beta 2 type. On mild tryptic cleavage it gives rise to a modified alpha 2 beta 2 form that has lost the tRNA(Phe) binding capacity but is still able to activate phenylalanine. In this paper are presented data concerning peptides released by this limited proteolytic conversion as well as those arising from exhaustive tryptic digestion of the truncated beta subunit. Each purified peptide was unambiguously assigned to a unique stretch of the beta subunit amino acid sequence that was recently determined via gene cloning and DNA sequencing. Together with earlier results from affinity labelling studies the present data show that the Lys 172-Ile 173 bond is the unique target of trypsin under mild conditions and that the N-terminal domain of each beta subunit (residues 1-172) contains the major tRNA(Phe) binding sites.     

Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerase chain reaction.

Taking advantage of sequence conservation of portions of the alpha, beta, and beta' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome. The polymerase chain reaction products were used as a probe to recover the genomic fragments encoding the beta subunit and the 5' portion of the beta' subunit from a library of cloned murine Chlamydia trachomatis DNA. Similar attempts to recover the alpha subunit were unsuccessful. Sequence analysis demonstrated that the beta subunit of RNA polymerase was located between genes encoding the L7/L12 ribosomal protein and the beta' subunit of RNA polymerase; this organization is reminiscent of the rpoBC operon of Escherichia coli. The C. trachomatis beta subunit overproduced in E. coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit. Although this polyclonal antibody specifically immunoprecipitated the beta subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme. Immunoblots with this antibody demonstrated that the beta subunit appeared early in infection.     

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.