Yeast phenylalanyl-tRNA Synthetase Research Articles

Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid.

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.

Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

As the number of reactions requiring biotransformation continues to grow, manipulating the enzyme substrate specificity becomes very important. Complementary to conventional enzyme engineering techniques based on only natural amino acids, we hypothesized that the site-specific incorporation of a non-natural amino acid in vivo into an enzyme can be used to re-design the active site for the altered substrate specificity. To test our hypothesis, we introduced the non-natural amino acid L-2-naphthylalanine (2Nal) into the active site of the model enzyme murine dihydrofolate reductase (mDHFR). We explored whether the substrate specificity of the enzyme could be switched from the good substrate dihydrofolate (DHF) to the poor substrate folate (FOL). We used two protein design programs (RosettaLigand and RosettaDesign) to calculate ligand docking and conformational stability, respectively, for the evaluation of multiples sites in the mDHFR. From the calculations, position 31 was predicted as an optimal 2Nal incorporation site. One mDHFR variant containing 2Nal at position 31 (mDHFR2Nal31) was expressed in the Escherichia coli expression host cells equipped with the engineered yeast phenylalanyl-tRNA and phenylalanyl-tRNA synthetase pair. As expected, the kinetic assays of purified mDHFR variant revealed that mDHFR2Nal31 has the enhanced binding affinity toward FOL and also exhibits 7.6-fold enhanced catalytic efficiency of FOL over DHF compared to mDHFRWT.

Tailoring the substrate specificity of yeast phenylalanyl-tRNA synthetase toward a phenylalanine analog using multiple-site-specific incorporation.

A yeast phenylalanyl-tRNA synthetase variant with T415G mutation (yPheRS (T415G)) was rationally designed to recognize various phenylalanine (Phe) analogs allowing site-specific incorporation into an amber site of a protein in E. coli. However, the relaxed substrate specificity of yPheRS (T415G) led to a significant tryptophan (Trp) misincorporation restricting the utility of yPheRS for biosynthesis of proteins containing a Phe analog. In order to obtain yPheRS variants with high substrate-specificity toward a Phe analog, we developed a general high-throughput screening method. This method uses fluorescence reduction of green fluorescence protein (GFP) upon efficient introduction of a Phe analog into multiple sites of GFP by breaking the degeneracy of the Phe codons. Combined use of positive and negative screenings of a yPheRS saturation library led to a yPheRS variant (yPheRS_naph) very selective toward 2-l-naphthylalanine (2Nal), a model Phe analog. The yPheRS_naph exhibited 6-fold higher relative activity toward 2Nal (vs Trp) in ATP-PPi exchange assays and led to high-fidelity incorporation of 2Nal into an amber site of murine dihydrofolate reductase in both minimal and rich media. These results successfully demonstrate that the high-throughput screening method developed can be used to evolve yPheRS to be very selective toward a Phe analog.

Site-Specific Incorporation of Tryptophan Analogues into Recombinant Proteins in Bacterial Cells

A designed yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) activates four tryptophan (Trp) analogues (6-chlorotryptophan (6ClW), 6-bromotryptophan (6BrW), 5-bromotryptophan (5BrW), and benzothienylalanine (BT)) that are not utilized by the endogenous E. coli translational apparatus. Introduction of yPheRS (T415G) and a mutant yeast phenylalanine amber suppressor tRNA (ytRNAPheCUA_UG) into an E. coli expression host allowed site-specific incorporation of three of these analogues (6ClW, 6BrW, and BT) into recombinant murine dihydrofolate reductase in response to amber stop codons with at least 98% fidelity. All three analogues were introduced at the Trp66 position in the chromophore of a cyan fluorescent protein variant (CFP6) to investigate the attendant changes in spectral properties. Each of the analogues caused blue shifts in the fluorescence emission and absorption maxima. The CFP6 variant bearing BT at position 66 exhibited an unusually large Stokes shift (56 nm). An expanded set of genetically encoded Trp analogues should enable the design of new proteins with novel spectral properties.

Design of a Bacterial Host for Site-Specific Incorporation of p-Bromophenylalanine into Recombinant Proteins

Introduction of a yeast suppressor tRNA (ytRNA(Phe)(CUA)) and a mutant yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) into an Escherichia coli expression host allows in vivo incorporation of phenylalanine analogues into recombinant proteins in response to amber stop codons. However, high-fidelity incorporation of non-natural amino acids is precluded in this system by mischarging of ytRNA(Phe)(CUA) with tryptophan (Trp) and lysine (Lys). Here we show that ytRNA(Phe)(CUA) and yPheRS can be redesigned to achieve high-fidelity amber codon suppression through delivery of p-bromophenylalanine (pBrF). Two strategies were used to reduce misincorporation of Trp and Lys. First, Lys misincorporation was eliminated by disruption of a Watson-Crick base pair between nucleotides 30 and 40 in ytRNA(Phe)(CUA). Loss of this base pair reduces mischarging by the E. coli lysyl-tRNA synthetase. Second, the binding site of yPheRS was redesigned to enhance specificity for pBrF. Specifically, we used the T415A variant, which exhibits 5-fold higher activity toward pBrF as compared to Trp in ATP-PP(i) exchange assays. Combining mutant ytRNA(Phe)(CUA) and yPheRS (T415A) allowed incorporation of pBrF into murine dihydrofolate reductase in response to an amber codon with at least 98% fidelity.

Breaking the degeneracy of the genetic code.

A mutant yeast phenylalanine transfer RNA (ytRNAPheAAA) containing a modified (AAA) anticodon was generated to explore the feasibility of breaking the degeneracy of the genetic code in Escherichia coli. By using an E. coli strain co-transformed with ytRNAPheAAA and a mutant yeast phenylalanyl-tRNA synthetase, we demonstrate efficient replacement of phenylalanine (Phe) by L-3-(2-naphthyl)alanine (Nal) at UUU, but not at UUC codons.

Anticodon domain methylated nucleosides of yeast tRNA(Phe) are significant recognition determinants in the binding of a phage display selected peptide.

The contributions of the natural modified nucleosides to RNA identity in protein/RNA interactions are not understood. We had demonstrated that 15 amino acid long peptides could be selected from a random phage display library using the criterion of binding to a modified, rather than unmodified, anticodon domain of yeast tRNA(Phe) (ASL(Phe)). Affinity and specificity of the selected peptides for the modified ASL(Phe) have been characterized by fluorescence spectroscopy of the peptides' tryptophans. One of the peptides selected, peptide t(F)2, exhibited the highest specificity and most significant affinity for ASL(Phe) modified with 2'-O-methylated cytidine-32 and guanosine-34 (Cm(32) and Gm(34)) and 5-methylated cytidine-40 (m(5)C(40)) (K(d) = 1.3 +/- 0.4 microM) and a doubly modified ASL(Phe)-Gm(34),m(5)C(40) and native yeast tRNA(Phe) (K(d) congruent with 2.3 and 3.8 microM, respectively) in comparison to that for the unmodified ASL(Phe) (K(d) = 70.1 +/- 12.3 microM). Affinity was reduced when a modification altered the ASL loop structure, and binding was negated by modifications that disfavored hairpin formation. Peptide t(F)2's higher affinity for the ASL(Phe)-Cm(32),Gm(34),m(5)C(40) hairpin and fluorescence resonance energy transfer from its tryptophan to the hypermodified wybutosine-37 in the native tRNA(Phe) placed the peptide across the anticodon loop and onto the 3'-side of the stem. Inhibition of purified yeast phenylalanyl-tRNA synthetase (FRS) catalyzed aminoacylation of cognate yeast tRNA(Phe) corroborated the peptide's binding to the anticodon domain. The phage-selected peptide t(F)2 has three of the four amino acids crucial to G(34) recognition by the beta-structure of the anticodon-binding domain of Thermus thermophilus FRS and exhibited circular dichroism spectral properties characteristic of beta-structure. Thus, modifications as simple as methylations contribute identity elements that a selected peptide specifically recognizes in binding synthetic and native tRNA and in inhibiting tRNA aminoacylation.

Pyrophosphate mediates the effect of certain tRNA mutations on aminoacylation of yeast tRNA(Phe).

The influence of pyrophosphate hydrolysis by inorganic pyrophosphatase on homologous aminoacylation of different yeast tRNA(Phe) mutants was studied. The addition of pyrophosphatase significantly improved the aminoacylation efficiency of tRNA(Phe) structural mutants as well as the mutant with substitution at position 20, while having no effect on the charge of wild-type tRNA(Phe). Aminoacylation of tRNA(Phe) anticodon and discriminator base (N(73)) mutants was not affected by pyrophosphatase. Activation of wild-type tRNA(Phe) transcript aminoacylation by inorganic pyrophosphatase was observed only at low Mg(2+) concentrations due to distortion of the tRNA(Phe) structure under these conditions. Our results demonstrate that pyrophosphate dissociation becomes a rate-limiting step of the reaction in yeast phenylalanyl-tRNA synthetase catalyzed aminoacylation of tRNA(Phe) variants with altered tertiary structure. A possible mechanism of pyrophosphate-mediated inhibition of tRNA mutants aminoacylation is discussed.

Crucial role of pyrophosphate in the aminoacylation of E. coli tRNA Phe by yeast phenylalanyl-tRNA synthetase

Rapid inactivation of the yeast phenylalanyl-tRNA synthetase in the course of aminoacylation of the heterologous E. coli tRNA Phe is observed. This inactivation occurs due to the formation of the tight complex of the enzyme with the pyrophosphate formed during the aminoacylation reaction. This complex is shown to be the normal intermediate of the reaction. Possible inactivation mechanism and correlation between structural differences of yeast and E. coli tRNAs Phe with the changes in the enzymatic mechanism of aminoacylation are discussed.

Recognition of yeast tRNA(Phe) by its cognate yeast phenylalanyl-tRNA synthetase: an analysis of specificity.

A kinetic analysis of aminoacylation of mutant yeast tRNA(Phe) transcripts by its cognate yeast phenylalanyl-tRNA synthetase (FRS) reveals five nucleotides in tRNA(Phe) as major recognition sites for FRS. The aminoacylation kinetics for two double mutants suggest that each of the five recognition sites contributes independently to kcat/KM. Measured kinetic values for the mutants presented here and those reported previously were then used to calculate the predicted kcat/KM of misacylation for a number of noncognate tRNAs. The predicted kcat/KM values are consistent with values measured by other investigators and thus support the five-nucleotide recognition model. The kcat/KM of misacylation for all known yeast tRNAs has been calculated on the basis of this model, and the specificity of FRS for tRNA(Phe) in yeast is discussed.

Recognition nucleotices for human phenylalanyl-tRNA synthetase

The specificity of the interaction between tRNAPhe and phenylalanyl-tRNA synthetase isolated from human placenta was investigated. Using yeast tRNAPhe transcripts with different point mutations it was shown that all the five recognition points for the yeast phenylalanyl-tRNA synthetase (G20, G34, A35, A36 and A73) are also important for the reaction catalyzed by the human enzyme. A set of mutations in nucleotides involved in tertiary interactions of tRNAPhe revealed that mutations which maintained the proper folding of the molecule had almost no influence on the efficiency of aminoacylation. The most striking difference between the yeast and human phenylalanyl-tRNA synthetases involved a mutation in the lower two base pairs of the anticodon stem. This mutation did not affect aminoacylation with the yeast enzyme, but greatly reduced activity with human phenylalanyl-tRNA synthetase.

Interaction of the tRNAPhe acceptor end with the synthetase involves a sequence common to yeast and Escherichia coli phenylalanyl-tRNA synthetases

Modified lysines resulting from the cross-linking of the 3' end of tRNA(Phe) to yeast phenylalanyl-tRNA synthetase (an enzyme with an alpha 2 beta 2 structure) have been characterized by sequencing the labeled chymotryptic peptides that were isolated by means of gel filtration and reversed-phase chromatography. The analysis showed that Lys131 and Lys436 in the alpha subunit are the target sites of periodate-oxidized tRNA(Phe). Mutant protein with a Lys----Asn substitution established that each lysine contributes to the binding of the tRNA but is not essential for catalysis. The major labeled lysine (K131) belongs to the sequence IALQDKL (residues 126-132), which shares three identities with the peptide sequence ADKL found around the tRNAox-labeled Lys61 in the large subunit of Escherichia coli phenylalanyl-tRNA synthetase [Hountondji, C., Schmitter, J. M., Beauvallet, C., & Blanquet, S. (1987) Biochemistry 26, 5433-5439].

Construction of aFRS1-FRS2operon encoding the structural genes for theαandβsubunits 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.

Reaction of nucleic acid bases with α-acetylenic esters: Part IV. Preparation of an alkylating derivative of tRNA Phe by conformation-specific chemical modification

The reaction of yeast tRNA Phe with methyl chlorotetrolate, ClCH 2-C≡C-COOCH 3, was studied. This reagent converts adenine and cytosine rings into derivatives in which an additional heterocycle bearing the alkylating chloromethyl group is fused to the original base; these derivatives can exist in two isomeric forms. Modified nucleosides of this type can be easily identified by reverse-phase HPLC. It was found that under native conditions, the modification of tRNA involves the anticodon loop and the 3′-end. The isomers of adenine derivatives formed in the anticodon loop were different from those formed in the 3′-end. It is suggested that the isomeric structure of the derivatives is related to the fine conformational differences between these two regions of tRNA Phe. Methyl chlorotetrolate could thus be used as a conformational probe of single-stranded nucleic acids. Preliminary assays showed that modified tRNA Phe binds irreversibly to yeast phenylalanyl-tRNA synthetase.

Structure and expression of the genes encoding the alpha and beta subunits of yeast phenylalanyl-tRNA synthetase.

The two genes FRS1 and FRS2 encoding, respectively, the large (alpha) and small (beta) subunits of cytoplasmic phenylalanyl-tRNA synthetase from bakers' yeast have been cloned and sequenced. The derived protein primary structures are confirmed by peptide sequences evenly distributed along the reading frames. These predict a subunit Mr of 67,347 for alpha and 57,433 for beta, in good agreement with earlier determinations carried out on the purified protein. These subunit sequences have been compared to those of Escherichia coli phenylalanyl-tRNA synthetase as well as to the small beta subunit of the corresponding yeast mitochondrial enzyme; limited but significant homology was found between the two alpha subunits on the one hand and between the three beta subunits on the other hand. The results suggest that these three enzymes, from E. coli, yeast cytoplasm, and yeast mitochondria, have strongly diverged from one another. The initiation sites of transcription have been determined for both yeast genes. Their 5'-upstream regions show no sequence similarities that would have indicated a coordinate control of gene expression at the transcriptional level. Measurements of steady-state levels of FRS-mRNAs in overproducing strains indicate that there is no restriction in mRNA synthesis. Therefore the control of gene expression, leading to a balanced synthesis of alpha and beta subunits, is likely to occur at the translational level.

Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro.

A recombinant plasmid was constructed with six synthetic DNA oligomers such that the DNA sequence corresponding to yeast tRNA(Phe) is flanked by a T7 promoter and a BstNI restriction site. Runoff transcription of the BstNI-digested plasmid with T7 RNA polymerase gives an unmodified tRNA of the expected sequence having correct 5' and 3' termini. This tRNA(Phe) transcript can be specifically aminoacylated by yeast phenylalanyl-tRNA synthetase and has a Km only 4-fold higher than that of the native yeast tRNA(Phe). The Km is independent of Mg2+ concentration, whereas the Vmax is very dependent on Mg2+ concentration. Comparison of the melting profiles of the native and the unmodified tRNA(Phe) at different Mg2+ concentrations suggests that the unmodified tRNA(Phe) has a less stable tertiary structure. Using one additional DNA oligomer, a mutant plasmid was constructed having a guanosine to thymidine change at position 20 in the tRNA gene. A decrease in Vmax/Km by a factor of 14 for aminoacylation of the mutant tRNA(Phe) transcript is observed.

Yeast phenylalanyl-tRNA synthetase: evidence for the formation of ADP by phosphorolysis of enzyme-bound aminoacyladenylate

ADP and Ap 3A are synthesized by the yeast phenylalanyl-tRNA synthetase, according to reaction pathways similar to the pyrophosphorolysis of the intermediate aminoacyladenylate or to the one leading to Ap 4A synthesis. The enzyme-bound phenylalanydenylate reacts with inorganic phosphate or ADP to yield, respectively, ADP or Ap 3A. The rate of synthesis is strongly stimulated by Zn 2+. This new phosphorolysis activity accounts for the complex pattern of bisnucleoside polyphosphate syntheses starting from ATP.

Covalent modification of phenylalanyl-tRNA synthetase with phenylalanine during the amino acid activation reaction catalyzed by the enzyme.

Yeast phenylalanyl-tRNA synthetase (PRS) is shown to undergo autoaminoacylation with phenylalanine under in vitro amino acid activation conditions. Phenylalanyl adenylate enzyme complex yields a covalent phenylalanyl isopeptide exclusively with the beta subunit of the alpha 2 beta 2 enzyme. Contrary to previously reported cases of autoaminoacylation of aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, the autoaminoacylation of PRS occurs under a specific set of conditions and results in the identification of only one labeled tryptic peptide on two types of high pressure liquid chromatography columns. The ability of PRS to undergo this covalent modification directly correlates with its ability to catalyze the synthesis of diadenosine 5',5"'-P1,P4-tetraphosphate from enzyme-bound phenylalanyl adenylate. Both reactions require the presence of low levels of zinc or cadmium and are inhibited by tRNAPhe or by low levels of low molecular weight thiols. Since diadenosine 5',5"'-P1,P4-tetraphosphate synthesis is known to be catalyzed in vivo in response to oxidation stress, it is also likely that the autoaminoacylation of phenylalanyl-tRNA synthetase may occur in vivo under a similar set of conditions. These reactions are thus not simply the result of accumulation of phenylalanyl adenylate and probably reflect conformational changes in the protein which are brought about by its interaction with zinc or cadmium.

Mechanism of activation of phenylalanine and synthesis of P1, P4-bis(5'-adenosyl) tetraphosphate by yeast phenylalanyl-tRNA synthetase.

The activation of L-phenylalanine by yeast phenylalanyl-tRNA synthetase using adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate is shown to proceed with inversion of configuration at P alpha of ATP. This observation taken together with the lack of positional isotope exchange when adenosine 5'-[beta,beta-18O2]triphosphate is incubated with the enzyme in the absence of phenylalanine and in the presence of the competitive inhibitor phenylalaninol indicates that activation of phenylalanine occurs by a direct "in-line" adenylyl-transfer reaction. In the presence of Zn2+, yeast phenylalanyl-tRNA synthetase also catalyzes the phenylalanine-dependent hydrolysis of ATP to AMP and the synthesis of P1,P4-bis(5'-adenosyl) tetraphosphate (Ap4A). With adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate, the formation of AMP and Ap4A is shown to occur with inversion and retention of configuration, respectively. It is concluded that phenylalanyl adenylate is an intermediate in both processes, Zn2+ promoting AMP formation by hydrolytic cleavage of the C-O bond and Ap4A formation by displacement at phosphorus of phenylalanine by ATP.

Specific replacement of functional groups of uridine-33 in yeast phenylalanine transfer ribonucleic acid.

Functional groups of the highly conserved uridine at position 33 in the anticodon loop of yeast tRNAPhe were altered by a synthetic protocol that replaces U-33 with any desired nucleotide and leaves all other nucleotides of the tRNA intact. The U-33-substituted tRNAs were prepared in an eight-step protocol that begins with partial cleavage of tRNAPhe at U-33 by ribonuclease A. By use of the combined half-molecules as substrate, U-33 was removed from the 5' half-molecule in three steps and then replaced by using RNA ligase to add the desired nucleoside 3',5'-bisphosphate. Each position 33 substituted 5' half-molecule was isolated and annealed to the original 3' half-molecule from the ribonuclease A digestion. The two halves were then rejoined in three steps to give a full-size tRNAPhe variant. This protocol should be applicable to other RNA molecules where a nucleotide substitution is desired at the 5' side of an available unique cleavage site. Seven substituted tRNAPheS containing uridine, pseudouridine, 3-methyluridine, 2'-O-methyluridine, cytidine, deoxycytidine, and purine riboside at position 33 were assayed for aminoacylation with yeast phenylalanyl-tRNA synthetase. Each of the seven tRNAs aminoacylated normally. Thus, unlike the adjacent guanine residue at position 34, U-33 is not involved in the interaction between yeast tRNAPhe and yeast phenylalanyl-tRNA synthetase.