Interactions between Escherichia coli arginyl-tRNA synthetase and its substrates

Interactions between Escherichia coli arginyl-tRNA synthetase and its substrates were extensively studied and distinctly demonstrated. Various approaches such as equilibrium dialysis, fluorescence titration, and substrate protection against heat inactivation of the enzyme were used for these studies. In the absence of other substrates, the equilibrium dissociation constants for arginine, ATP, and the cognate tRNA were about 70 microM, 0.85 mM, and 0.45 microM, respectively, at pH 7.5, in Tris buffer. The binding of arginine to the enzyme was affected neither by the presence of tRNA nor by the presence of ATP but was considerably enhanced when ATP and tRNA were both present at saturating concentrations. The dissociation constant in this case (about 16 microM) was very close to the Km (12 microM) for arginine during aminoacylation. The binding of ATP (the equilibrium dissociation constant KD approximately 0.85 mM) was not affected by the presence of arginine but was depressed in the presence of tRNA (KD became 3 mM). Arginyl-tRNA showed a dissociation constant of (4-5) X 10(-7) M which was not affected by the presence of a single other substrate. Possible explanations for the high Km for tRNA in the aminoacylation are discussed. Our results indicated pronounced interactions between substrates mediated by the enzyme under catalytic conditions. Periodate oxidation did not alter the tRNA binding to the enzyme. The oxidized tRNA still afforded protection against heat inactivation of the enzyme.     

Cloning and characterization of the gene for Escherichia coli seryl-tRNA synthetase.

Seryl-tRNA synthetase is the gene product of the serS locus in Escherichia coli. Its gene has been cloned by complementation of a serS temperature sensitive mutant K28 with an E. coli gene bank DNA. The resulting clones overexpress seryl-tRNA synthetase by a factor greater than 50 and more than 6% of the total cellular protein corresponds to the enzyme. The DNA sequence of the complete coding region and the 5'- and 3' untranslated regions was determined. Protein sequence comparison of SerRS with all available aminoacyl-tRNA synthetase sequences revealed some regions of significant homology particularly with the isoleucyl- and phenylalanyl-tRNA synthetases from E. coli.     

Structure-function relationship of arginyl-tRNA synthetase from Escherichia coli: isolation and characterization of the argS mutation MA5002.

The Escherichia coli K12 argS MA5002 mutant appears to have a functionally altered arginyl-tRNA synthetase (ArgRS). The gene coding for this enzyme was isolated from E. coli genomic DNA using the PCR procedure and inserted into a pUC18 multicopy vector. Sequencing revealed that it differs from the wildtype ArgRS structural gene only by one mutation: a replacement of a C by an A residue which results in substitution of an arginine by a serine at position 134, located two residues downstream from the HVGH consensus sequence. As compared to the genomic enzyme level, this recombinant vector, containing the mutated gene, produces in E. coli JM103, about 100 times as much modified ArgRS. This enzyme was obtained nearly pure after only two chromatographic steps; it exhibits a 4-6 times as low activity and a 5 times as high Km value for ATP as the wildtype enzyme in the aminoacylation and ATP-PPi reactions; Km values for arginine and tRNAArg remained unaltered. The position of this mutation and its effect on enzymatic properties suggest the implication of arginine 134 in ATP binding as well as in the activation catalytic process.     

Crystallization and preliminary X-ray diffraction analysis of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA Synthetase, a class I aminoacyl tRNA synthetase playing a crucial role in protein biosynthesis, has been crystallized for the first time. Polyethylene glycol (PEG) was used as a precipitant, and the crystallization proceeded at pH 6.5. These single crystals diffracted to 2.8 A with a rotating anode X-ray source and R-axis IIc image plate detector. They have an orthorhombic space group P2(1)2(1)2 with unit cell parameters of a = 251.51 A, b = 53.12 A, and c = 52.35 A. A complete native data set has been collected at 3.1 A resolution for these crystals.     

Cloning and characterization of the Escherichia coli gene coding for alkaline phosphatase.

The Escherichia coli structural gene for alkaline phosphatase, phoA, and a promoter-like mutant of phoA, called pho-1003(Bin) phoA+, were cloned by using plasmid vectors. Initially, these genes were cloned on deoxyribonucleic acid fragments of 28.9 kilobases (kb). Subsequently, they were subcloned on fragments and 4.8 and then 2.7 kilobases. A restriction map was developed, and phoA was localized to a 1.7-kb region. The promoter end of the gene was inferred by its proximity to another gene cloned on the same deoxyribonucleic acid fragment, proC. The stability of the largest plasmid (33.3 kb) was found to be recA dependent, although the subcloned plasmids were stable in a recA+ strain. Synthesis of alkaline phosphatase directed by the phoA+ and pho-1003(Bin) phoA+ plasmids in a phoA deletion strain was assayed under repressing and derepressing levels of phosphate. These data were compared with the copy numbers of the plasmids. It was found that synthesis of alkaline phosphatase was tightly regulated, even under derepressing conditions: a copy number of 17 enabled cells to synthesize only about twofold more enzyme than did cells with 1 chromosomal copy of phoA+. Enzyme levels were also compared for cells containing pho-1003(Bin) phoA+ and phoA+.     

Effect of cysteine residues on the activity of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA synthetase (ArgRS) from Escherichia coli (E. coli) contains four cysteine residues. In this study, the role of cysteine residues in the enzyme has been investigated by chemical modification and site-directed mutagenesis. Titration of sulfhydryl groups in ArgRS by 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) suggested that a disulfide bond was not formed in the enzyme and that, in the native condition, two DTNB-sensitive cysteine residues were located on the surface of ArgRS, while the other two were buried inside. Chemical modification of the native enzyme by iodoacetamide (IAA) affected only one DTNB-sensitive cysteine residue and resulted in 50% loss of enzyme activity, while modification by N-ethylmeimide (NEM) affected two DTNB-sensitive residues and caused a complete loss of activity. These results, when combined with substrate protection experiments, suggested that at least the two cysteine residues located on the surface of the molecule were directly involved in substrates binding and catalysis. However, changing Cys to Ala only resulted in slight loss of enzymatic activity and substrate binding, suggesting that these four cysteine residues in E. coli ArgRS were not essential to the enzymatic activity. Moreover, modifications of the mutant enzymes indicated that the two DTNB- and NEM-sensitive residues were Cys(320) and Cys(537) and the IAA-sensitive was Cys(320). Our study suggested that inactivation of E. coli ArgRS by sulfhydryl reagents is a result of steric hindrance in the enzyme.     

Map location of arginyl-tRNA synthetase mutations in Escherichia coli K-12

Summary Mutants of Escherichia coli K-12 previously isolated in the authors' laboratory have reduced arginyl-tRNA synthetase activity. The mutants fall into two classes. All mutants grow slowly on arginine-free medium. On arginine-supplemented medium some mutants grow at a normal rate (Class I) while others still grow slowly (Class II). Matings were performed to located a Class I and a Class II mutation on the E. coli chromosome map, and on the basis of our results we have assigned both to one locus, argS.     

Escherichia coli glutaminyl-tRNA synthetase. I. Isolation and DNA sequence of the glnS gene

We have isolated a lambda-transducing phage carrying the gene (glnS) for Escherichia coli glutaminyl-tRNA synthetase. The location of the glnS gene within the 13.5-kilobase E. coli DNA transducing fragment was determined by genetic means. The glnS gene was recloned into plasmid pBR322 and its nucleotide sequence was established. The DNA sequence translates to a protein of 550 amino acids.     

Isolation and characterization of the Escherichia coli mutH gene product

The Escherichia coli mutH gene product has been isolated in near homogeneous form using an in vitro complementation assay for DNA mismatch correction (Lu, A.-L., Clark, S., and Modrich, P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4639-4643) which is dependent on mutH function. The protein has a subunit Mr of 25,000, and purified preparations contain a Mg2+-dependent endonuclease activity which cleaves 5' to the dG of d(GATC) sequences to generate 5'-phosphoryl and 3'-hydroxyl termini. Symmetrically methylated d(GATC) sites are resistant to the endonuclease, hemimethylated sequences are cleaved on the unmethylated strand, and unmethylated d(GATC) sites are usually subject to scission on only one DNA strand. Although this endonuclease activity is extremely weak (less than 1 scission/h/mutH monomer equivalent) and cleavage at a d(GATC) site does not depend on the presence of a mismatched base pair within the DNA substrate, the activity does not appear to be a contaminant of mutH preparations. d(GATC) endonuclease activity and mutH complementing activity co-purify through multiple column steps without change in relative specific activities, and both activities co-electrophorese under native conditions. These findings suggest that the mutH product functions at the strand discrimination stage of mismatch correction and that this stage of the reaction involves scission of the unmethylated DNA strand.     

Isolation and characterization of the Escherichia coli mutL gene product

The Escherichia coli mutL gene product has been purified to near homogeneity from an overproducing clone. The mutL locus encodes a polypeptide of 70,000 daltons as determined by denaturing gel electrophoresis. The native molecular weight of MutL protein as calculated from the sedimentation coefficient of 5.5 S and Stokes radius of 61 A is 139,000 daltons, indicating that MutL exists as a dimer in solution. In addition to its ability to complement methyl-directed DNA mismatch repair in mutL-deficient cell-free extracts, DNase I protection experiments demonstrate that the purified MutL protein interacts with the MutS-heteroduplex DNA complex in the presence of ATP.     

Isolation and characterization of the yeast gene coding for the alpha subunit of mitochondrial phenylalanyl-tRNA synthetase

The respiratory defect of pet mutants of Saccharomyces cerevisiae assigned to complementation group G120 has been ascribed to their inability to acylate the mitochondrial phenylalanyl tRNA. A fragment of wild type yeast genomic DNA capable of complementing the genetic lesion of G120 mutants has been cloned by transformation with a yeast genomic recombinant library of a representative mutant from this complementation group. The gene designated as MSF1 has been subcloned on a 2.2-kilobase pair fragment and its nucleotide sequence determined. The predicted protein product of MSF1 has a molecular weight of 55,314 and has several domains of high primary sequence homology to the alpha subunit of the Escherichia coli phenylalanyl-tRNA synthetase. Based on the phenotype of G120 mutants and the homology to the bacterial protein, MSF1 is proposed to code for the alpha subunit of yeast mitochondrial phenylalanyl-tRNA synthetase. Disruption of the chromosomal copy of MSF1 in the respiratory-competent haploid strain W303-1B induces a phenotype similar to G120 mutants but does not affect cell viability, indicating that the cytoplasmic phenylalanyl-tRNA synthetase of yeast is encoded by a separate gene. Although the E. coli and yeast mitochondrial aminoacyl-tRNA synthetases are sufficiently similar in their primary sequences to suggest a common evolutionary origin, they have undergone significant changes as evidenced by the low homology in some regions of the polypeptide chains and the presence in the mitochondrial enzyme of two domains that are lacking in the bacterial phenylalanyl-tRNA synthetase.     

Functional expression in Escherichia coli of the Haemophilus influenzae gene coding for selenocysteine-containing selenophosphate synthetase

The selenophosphate synthetases from several organisms contain a selenocysteine residue in their active site where the Escherichia coli enzyme contains a cysteine. The synthesis of these enzymes, therefore, depends on their own reaction product. To analyse how this self-dependence is correlated with the selenium status, e.g. after recovery from severe selenium starvation, we expressed the gene for the selenocysteine-containing selenophosphate synthetase from Haemophilus influenzae (selD _HI) in an E. coliΔselD strain. Gene selD _HI gave rise to a selenium-containing gene product and also supported – via its activity – the formation of E. coli selenoproteins. The results provide evidence either for the suppression of the UGA_Sec codon with the insertion of an amino acid allowing the formation of a functional product or for a bypass of the selenophosphate requirement. We also show that the selenocysteine synthesis and the insertion systems of the two organisms are fully compatible despite conspicuous differences in the mRNA recognition motif. Received: 8 July 1997 / Accepted: 3 September 1997     

Studies on arginyl-tRNA synthetase from Escherichia coli B. Dual role of metals in enzyme catalysis.

Studies carried out in arginyl-tRNA synthetase from Escherichia coli indicate that metals may have two functional roles in the catalytic mechanism. Complete metal activation is observed when MgCl2, MnCl2, CoCl2, or FeCl2 is present at a concentration (5.0 mM) in excess of the total ATP concentration (2.0 mM). When CaCl2 is substituted for MgCl2, activity is not observed unless a small amount (0.1 mM) of MgCl2, MnCl2, CoCl2, FeCl2, or ZnCl2 (unable to produce activity alone at 5.0 mM) is added. A model, based on kinetic data, is proposed in which the enzyme possesses a site for free metal, which, when filled, lowers the Km for all three substrates (arginine, tRNAArg, and metal-ATP) and increases the Vmax of the reaction.     

Isolation and characterization of independently folding regions of the beta chain of Escherichia coli tryptophan synthetase.

It had been reported previously that the beta2 subunit of Escherichia coli tryptophan synthetase [L-serinehydrolyase (adding indole) EC 4.2.1.20] can be cleaved by trypsin into a nearly functional dimeric protein, the monomer of which consists of two large, nonoverlapping, polypeptide fragments. In the present paper, it is shown that these fragments can be separated after denaturation. Upon removal of the denaturing agent, the isolated fragments spontaneously refold into conformation, which, by various physical-chemical criteria, are shown to approximate the conformations of the corresponding fragments associated within the native protein. Furthermore, it is demonstrated that, upon mixing, these renatured fragments reassociate to form the renatured nicked protein which, by all the physical and functional criteria used, is indistinguishable from the native nicked protein. These results are taken as strong evidence that the isolated fragments can be considered as independently folding regions corresponding to intermediates in the folding of the intact protein.     

Cloning of the gene for Escherichia coli glutamyl-tRNA synthetase

The structural gene for the glutamyl-tRNA synthetase of Escherichia coli has been cloned in E. coli strain JP1449, a thermosensitive mutant altered in this enzyme. Ampicillin-resistant and tetracycline-sensitive thermoresistant colonies were selected following the transformation of JP1449 by a bank of hybrid plasmids containing fragments from a partial Sau3A digest of chromosomal DNA inserted into the BamHI site of pBR322. One of the selected clones, HS7611, has a level of glutamyl-tRNA synthetase activity more than 20 times higher than that of a wild-type strain. The overproduced enzyme has the same molecular weight and is as thermostable as that of a wild-type strain, indicating that the complete structural gene is present in the insert. These characteristics were lost by curing this clone of its plasmid with acridine orange, and were transferred with high efficiency to the mutant strain JP1449 by transformation with the purified plasmid. A physical map of the plasmid, which contains an insert of about 2.7 kb in length, is presented.     

Shifted positioning of the anticodon nucleotide residues of amber suppressor tRNA species by Escherichia coli arginyl-tRNA synthetase.

Cytidine in the anticodon second position (position 35) and G or U in position 36 of tRNAArg are required for aminoacylation by arginyl-tRNA synthetase (ArgRS) from Escherichia coli. Nevertheless, an arginine-accepting amber suppressor tRNA with a CUA anticodon (FTOR1Delta26) exhibits suppression activity in vivo [McClain, W.H. & Foss, K. (1988) Science, 241, 1804-1807]. By an in vitro kinetic study with mutagenized tRNAs, we showed that the arginylation of FTOR1Delta26 involves C34 and U35, and that U35 can be replaced by G without affecting the activity. Thus, the positioning of the essential nucleotides for the arginylation is shifted to the 5' side, by one residue, in the suppressor tRNAArg. We found that the shifted positioning does not depend on the tRNA sequence outside the anticodon. Furthermore, by a genetic method, we isolated a mutant ArgRS that aminoacylates FTOR1Delta26 more efficiently than the wild-type ArgRS. The isolated mutant has mutations at two nonsurface amino-acid residues that interact with each other near the anticodon-binding site.