Functional mutants of phenylalanine transfer RNA from Escherichia coli.

The gene pheV from Escherichia coli, coding for tRNAPhe and carried on a plasmid, has been mutagenised with hydroxylamine. Mutants in the structural gene have been identified using two criteria: (i) de-attenuation of beta-galactosidase expression, while under the control of the attenuator region of the pheS,T operon by means of an operon fusion; (ii) loss of ability to complement thermosensitivity of a mutant Phe-tRNA synthetase. Mutants showing de-attenuation were sequenced and two nucleotide changes identified: G44----A44 (found five times) and m7G46----A46 (found once). Sequencing of mutants that lost complementation identified two further tRNA mutants, C2---U2 and G15----A15; the mutant m7G46----A46 was also re-isolated by this criterion. Three of the mutants involve bases implicated in tertiary rather than secondary structure hydrogen bonding. One hypothesis for the mechanism of de-attenuation is that mutant tRNAPhe molecules compete with the wild-type tRNAPhe on the ribosome but are inefficient at some step in the elongation process.     

Mutants affecting tRNA(Phe) from Escherichia coli. Studies of the suppression of thermosensitive phenylalanyl-tRNA synthetase

Four mutants of pheV, a gene coding for tRNA(Phe) in Escherichia coli, share the characteristic that when carried in the plasmid pBR322, they lose the capacity of wild-type pheV to complement the thermosensitive defect in a mutant of phenylalanyl-tRNA synthetase. One of these mutants, leading to the change C2----U2 in tRNA(Phe), is expressed about 10-fold lower in transformed cells than wild-type pheV. This mutant, unlike the remaining three (G15----A15, G44----A44, m7G46----A46), can recover the capacity to complement thermosensitivity when carried in a plasmid of higher copy number. The other three mutants, even when expressed at a similar level, remain unable to complement thermosensitivity. A study of charging kinetics suggests that the loss of complementation associated with these mutants is due to an altered interaction with phenylalanyl-tRNA synthetase. The mutant gene pheV (U2), when carried in pBR322, can also recover the capacity to complement thermosensitivity through a second-site mutation outside the tRNA structural gene, in the discriminator region. This mutation, C(-6)----T(-6), restores expression of the mutant U2 to about the level of wild-type tRNA(Phe).     

Defective regulation of the phenylalanine biosynthetic operon in mutants of the phenylalanyl-tRNA synthetase operon.

Among mutants of Escherichia coli resistant to p-fluorophenylalanine (PFP) were some with constitutive expression of the phenylalanine biosynthetic operon (the pheA operon). This operon is repressed in the wild type by phenylalanine. The mutation in three of these mutants mapped in the aroH-aroD region of the E. coli chromosome at 37 min. A plasmid bearing wild-type DNA from this region restored p-fluorophenylalanine sensitivity and wild-type repression of the pheA operon. Analysis of subclones of this plasmid and comparison of its restriction map with published maps indicated that the mutations affecting regulation of the pheA operon lie in the structural genes for phenylalanyl-tRNA synthetase, pheST, probably in pheS. Thus, the pheST operon has a role in the regulation of phenylalanine biosynthesis, the most likely being that wild-type phenylalanyl-tRNA synthetase maintains a sufficient intracellular concentration of Phe-tRNA(Phe) for attenuation of the pheA operon in the presence of phenylalanine. A revised gene order for the 37-min region of the chromosome is reported. Read clockwise, the order is aroD, aroH, pheT, and pheS.     

The pheR gene of Escherichia coli encodes tRNA(Phe), not a repressor protein

Nucleotide sequence analysis and transposon 5 (Tn5) insertional mutagenesis indicate that the Escherichia coli gene pheR encodes tRNA(Phe) and not a repressor protein as previously reported. The coding region of pheR is identical to that of three other cloned tRNA(Phe) genes, pheU, pheV, and pheW. Multicopy plasmids carrying pheR, like those carrying pheU, pheV, or pheW, complement a temperature-sensitive lesion in the gene for the alpha-subunit of phenylalanyl-tRNA synthetase (pheS). The nucleotide sequences of the 5'-flanking DNA of pheR, pheU, and pheW are almost identical but are quite different from the same region of pheV. By comparison with pheV, which has two tandem promoters, pheR was found to have a single promoter. The expression of pheA (encoding chorismate mutase/prephenate dehydratase) in strains carrying the pheR374 allele was decreased to similar extents by multicopy plasmids containing either pheR or pheV. It is proposed that this decrease in pheA expression and the increase in expression of pheA previously reported for chromosomal pheR mutants are both mediated through the attenuation control mechanism that regulates pheA.     

Control of phenylalanyl-tRNA synthetase genetic expression. Site-directed mutagenesis of the pheS, T operon regulatory region in vitro

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli strongly suggested that the pheS, T operon was regulated by a phenylalanine-mediated attenuation mechanism. To investigate the functions of the different segments composing the pheS, T attenuator site, a series of insertion, deletion and point mutations in the pheS, T leader region have been constructed in vitro on a recombinant M13 phage. The effects of these alterations on the regulation of the operon were measured after transferring each mutation onto a lambda phage carrying a pheS, T-lacZ fusion. The behaviours of the various mutants agree with the predictions of the attenuation model. The role of the antiterminator (2-3 pairing) as competitor of the terminator (3-4 pairing) is demonstrated by several mutations affecting the stability of the 2-3 base-pairing. The existence of deletions and point mutations in the 3-4 base-pairing shows that the terminator is essential for both expression level and regulation of the operon. Mutations in the translation initiation site of the leader peptide show that the expression of the leader peptide is essential for attenuation control. However, alteration of the translation initiation rate of the leader peptide derepresses the pheS, T operon, which is the opposite of what is observed with the trp operon. This difference is explained in terms of different translation initiation efficiencies of the leader peptides. Finally, insertion mutations, increasing gradually the distance between the leader peptide stop codon and the first strand of the antiterminator, derepress the pheS, T operon and show that formation of the antiterminator structure is under the control of the translation of the leader peptide.     

Phenylalanyl-tRNA synthetase of Escherichia coli K 10. Multiple enzyme-aminoacyl-tRNA complexes as a consequence of substrate specificity.

The interaction between Phe-tRNA(Phe) or other acyl-tRNA derivatives thereof and phenylalanyl-tRNA synthetase of Escherichia coli K 10 has been investigated by nonequilibrium dialysis, by fluorescence titration in the presence of 2-p-toluidinylnaphthalene-6-sulfonate, by the kinetics of the aminoacylation of tRNA(Phe), and by the kinetics of the catalytic hydrolysis of Phe-tRNA(Phe). Phe-tRNA(Phe), or derivatives thereof, forms two types of complexes with the synthetase. One type involves the attachment of the phenylalanyl moiety to the phenylalanine-specific site of the enzyme, and the other type, to the tRNA(Phe)-specific binding site. They resemble alternative modes of a destabilized enzyme-product complex and are predicted on the basis of thermodynamic considerations. The two modes of binding of acyl-tRNA compete with each other. The attachment of Phe-tRNA(Phe) to the phenylalanine-specific site dominates. At equilibrium, this complex is present at a fourfold higher concentration than the other type of complex. The HNO2 deaminated Phe-tRNA(Phe) binds exclusively to the site specific for L-phenylalanine. On the contrary, Ile-tRNA(Phe) adds at 94.1% to the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) with this site leads to enzymatic hydrolysis into L-phenylalanine and tRNA(Phe). The complex involving the phenylalanine-specific site is hydrolytically unproductive. L-Phenylalanine acts as an activator of the hydrolysis by occupying the amino acid specific site and by shifting the equilibrium between the complexes toward the binding ot Phe-tRNA(Phe) at the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) at the phenylalanine-specific site does not interfere sterically with the binding of free tRNA(Phe). The sequential addition of free and aminoacylated tRNA(Phe) exhibits negative cooperativity. Such a mechanism could help to expel the product from the enzyme.     

Regulation of E.coli phenylalanyl-tRNA synthetase operon in vivo

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNAPhe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.     

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.     

Phenylalanyl-tRNA synthetase editing defects result in efficient mistranslation of phenylalanine codons as tyrosine

Translational quality control is monitored at several steps, including substrate selection by aminoacyl-tRNA synthetases (aaRSs), and discrimination of aminoacyl-tRNAs by elongation factor Tu (EF-Tu) and the ribosome. Phenylalanyl-tRNA synthetase (PheRS) misactivates Tyr but is able to correct the mistake using a proofreading activity named editing. Previously we found that overproduction of editing-defective PheRS resulted in Tyr incorporation at Phe-encoded positions in vivo, although the misreading efficiency could not be estimated. This raised the question as to whether or not EF-Tu and the ribosome provide further proofreading mechanisms to prevent mistranslation of Phe codons by Tyr. Here we show that, after evading editing by PheRS, Tyr-tRNA(Phe) is recognized by EF-Tu as efficiently as the cognate Phe-tRNA(Phe). Kinetic decoding studies using full-length Tyr-tRNA(Phe) and Phe-tRNA(Phe), as well as a poly(U)-directed polyTyr/polyPhe synthesis assay, indicate that the ribosome lacks discrimination between Tyr-tRNA(Phe) and Phe-tRNA(Phe). Taken together, these data suggest that PheRS editing is the major proofreading step that prevents infiltration of Tyr into Phe codons during translation.     

Post-transfer editing in vitro and in vivo by the beta subunit of phenylalanyl-tRNA synthetase

Translation of the genetic code requires attachment of tRNAs to their cognate amino acids. Errors during amino-acid activation and tRNA esterification are corrected by aminoacyl-tRNA synthetase-catalyzed editing reactions, as extensively described for aliphatic amino acids. The contribution of editing to aromatic amino-acid discrimination is less well understood. We show that phenylalanyl-tRNA synthetase misactivates tyrosine and that it subsequently corrects such errors through hydrolysis of tyrosyl-adenylate and Tyr-tRNA(Phe). Structural modeling combined with an in vivo genetic screen identified the editing site in the B3/B4 domain of the beta subunit, 40 angstroms from the active site in the alpha subunit. Replacements of residues within the editing site had no effect on Phe-tRNA(Phe) synthesis, but abolished hydrolysis of Tyr-tRNA(Phe) in vitro. Expression of the corresponding mutants in Escherichia coli significantly slowed growth, and changed the activity of a recoded beta-galactosidase variant by misincorporating tyrosine in place of phenylalanine. This loss in aromatic amino-acid discrimination in vivo revealed that editing by phenylalanyl-tRNA synthetase is essential for faithful translation of the genetic code.     

Evidence that there are only two tRNA(Phe) genes in Escherichia coli.

pheV, one of the genes that code for tRNA(Phe), was deleted from the chromosome of a strain of Escherichia coli K-12. As a consequence of this mutation, expression of pheA, the gene for chorismate mutase P-prephenate dehydratase, the first enzyme in the terminal pathway of phenylalanine biosynthesis, was derepressed. Similar derepression of pheA has been reported in pheR mutants of E. coli K-12 (J. Gowrishankar and J. Pittard, J. Bacteriol. 150:1130-1137, 1982). Attempts to introduce a pheR mutation into the delta pheV strain failed under circumstances suggesting that this combination of mutations is lethal. Southern blot analysis of pheV+ and delta pheV strains indicated that there are only two tRNA(Phe) genes in E. coli. It is recommended that the names pheU and pheV be retained for these genes.     

Regulation of E. coli phenylalanyl-tRNA synthetase operon in vivo

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNA^Phe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.     

Open reading frames in the control regions of the phenylalanyl-tRNA synthetase operon of E. coli

The pheST operon codes for the two subunits of phenylalanyl-tRNA synthetase and it expression is controlled by attenuation in a way similar to many amino acid biosynthetic operons. The nucleotide sequence of the control regions of the operon indicates the presence of several open reading frames besides that of the leader peptide. One of these open reading frames, called the alternative leader peptide, starts at about the same place as the leader peptide and ends after the terminator of the attenuator. Another open reading frame, called the terminator peptide, starts after the terminator and covers about half the distance to pheS, the first structural gene of the operon. The present report shows that, in fact, the only open reading frame to be translated efficiently is the leader peptide itself. The alternative leader peptide and the terminator peptide are both translated at a negligible rate.     

Threonyl-Transfer Ribonucleic Acid Synthetase from Escherichia coli: Subunit Structure and Genetic Analysis of the Structural Gene by Means of a Mutated Enzyme and of a Specialized Transducing Lambda Bacteriophage

Threonyl-transfer ribonucleic acid synthetase (ThrRS) has been purified from a strain of Escherichia coli that shows a ninefold overproduction of this enzyme. Determination of the molecular weight of the purified, native enzyme by gel chromatography and by polyacrylamide gel electrophoresis at different gel concentrations yielded apparent molecular weight values of 150,000 and 161,000, respectively. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band of 76,000-dalton size. From these results an alpha(2) subunit structure can be inferred. A mutant with a structurally altered ThrRS, which had been obtained by selection for resistance against the antibiotic borrelidin, was used to map the position of the ThrRS structural gene (thrS) by P1 transductions. It was found that thrS is located in the immediate neighborhood of pheS and pheT, which are the structural genes for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase, the gene order being aroD-pheT-pheS-thrS. A lambda phage that was previously shown to specifically transduce pheS, pheT, and also the structural gene for the translation initiation factor IF3 can complement the defect of the altered ThrRS of the borrelidin-resistant strain. This phage also stimulates the synthesis of the 76,000, molecular-weight polypeptide of ThrRS in ultraviolet light-irradiated. E. coli cells. These results indicate that the genes for ThrRS, alpha and beta subunits of phenylalanyl-tRNA synthetase, and initiation factor IF3 are immediately adjacent on the E. coli chromosome.     

Kinetic analysis of an E.coli phenylalanine-tRNA synthetase mutant.

A mutation in the pheS gene, encoding phenylalanyl-tRNA synthetase, in E. coli NP37 confers temperature-sensitivity on the organism. A five-fold increase in tRNA(phe) levels complements the mutation. Analysis of the kinetic properties of the mutant enzyme indicates that the KM is 20-fold higher than the wild-type and the dissociation constant of the tRNA(phe)-synthetase complex for the mutant is at least 10-fold higher. These results indicate that the mutation in E. coli NP37 directly affects the tRNA(phe) binding site on the cognate synthetase.