Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli

Respiratory deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G59 are pleiotropically deficient in respiratory chain components and in mitochondrial ATPase. This phenotype has been shown to be a consequence of mutations in a nuclear gene coding for mitochondrial leucyl-tRNA synthetase. The structural gene (MSL1) coding for the mitochondrial enzyme has been cloned by transformation of two different G59 mutants with genomic libraries of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein of 894 residues with a molecular weight of 101,936. The amino-terminal sequence (30-40 residues) has a large percentage of basic and hydroxylated residues suggestive of a mitochondrial import signal. The cloned MSL1 gene was used to construct a strain in which 1 kb of the coding sequence was deleted and substituted with the yeast LEU2 gene. Mitochondrial extracts obtained from the mutant carrying the disrupted MSL1::LEU2 allele did not catalyze acylation of mitochondrial leucyl-tRNA even though other tRNAs were normally charged. These results confirmed the correct identification of MSL1 as the structural gene for mitochondrial leucyl-tRNA synthetase. Mutations in MSL1 affect the ability of yeast to grow on nonfermentable substrates but are not lethal indicating that the cytoplasmic leucyl-tRNA synthetase is encoded by a different gene. The primary sequence of yeast mitochondrial leucyl-tRNA synthetase has been compared to other bacterial and eukaryotic synthetases. Significant homology has been found between the yeast enzyme and the methionyl- and isoleucyl-tRNA synthetases of Escherichia coli. The most striking primary sequence homology occurs in the amino-terminal regions of the three proteins encompassing some 150 residues. Several smaller domains in the more internal regions of the polypeptide chains, however, also exhibit homology. These observations have been interpreted to indicate that the three synthetases may represent a related subset of enzymes originating from a common ancestral gene.     

Characterization of MSM1, the structural gene for yeast mitochondrial methionyl-tRNA synthetase

Respiratory-deficient mutants of Saccharomyces cerevisiae assigned to pet complementation group G72 are impaired in mitochondrial protein synthesis. The loss of this activity has been correlated with the inability of the mutants to acylate the two methionyl-tRNAs of yeast mitochondria. A nuclear gene (MSM1) capable of complementing the respiratory deficiency has been cloned by transformation of the G72 mutant C122/U3 with a yeast genomic library. In situ disruption of the MSM1 gene in a wild-type haploid strain of yeast induces a respiratory-deficient phenotype but does not affect the ability of the mutant to grow on fermentable substrates indicating that the product of MSM1 functions only in mitochondrial protein synthesis. Mitochondrial extracts prepared from the mutant with the disrupted copy of MSM1 were found to be defective in acylation of the two mitochondrial methionyl-tRNAs thereby confirming the identity of MSM1 as the structural gene for the mitochondrial methionyl-tRNA synthetase. The sequence of the protein encoded by MSM1 is similar to the Escherichia coli and yeast cytoplasmic methionyl-tRNA synthetases. Based on the primary-sequence similarities of the three proteins, the mitochondrial enzyme appears to be more related to the bacterial than to the yeast cytoplasmic methionyl-tRNA synthetase.     

Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.

The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria. The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression. FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype. The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase. The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN. This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway. A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S. cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.     

Bacillus subtilis phenylalanyl-tRNA synthetase genes: cloning and expression in Escherichia coli and B. subtilis.

The genes that encode the two subunits of Bacillus subtilis phenylalanyl-tRNA synthetase were cloned from alpha lambda library of chromosomal B. subtilis DNA by specific complementation of a thermosensitive Escherichia coli pheS mutation. Both genes (we named them pheS and pheT, analogous to the corresponding genes of E. coli) are carried by a 6.6-kilobase-pair PstI fragment which also complements E. coli pheT mutations. This fragment directs the synthesis of two proteins identical in size to the purified alpha and beta subunits of the phenylalanyl-tRNA synthetase of B. subtilis with Mrs of 42,000 and 97,000, respectively. A recombinant shuttle plasmid carrying the genes caused 10-fold overproduction of functional phenylalanyl-tRNA synthetase in B. subtilis.     

Isolation of the structural genes for the alpha and beta subunits of the mitochondrial ATPase from the fission yeast Schizosaccharomyces pombe

The structural genes for the two major subunits of the mitochondrial ATPase were isolated among genomic clones from the yeast Schizosaccharomyces pombe by transformation and complementation of mutants unable to grow on glycerol and lacking either the alpha or the beta subunits. The plasmid pMa1 containing a 2.3-kilobase genomic insert transformed the mutant A23-13 lacking a detectable alpha subunit. The transformant grew on glycerol and contained an alpha subunit of normal electrophoretic mobility. The plasmid pMa2 containing a 5.4-kilobase genomic insert transformed the mutant B59-1 lacking the beta subunit. The transformant grew on glycerol and contained a beta subunit of normal mobility. The structural gene for the beta ATPase subunit for the fission yeast S. pombe was localized within the pMa2 insert by hybridization to a probe containing the beta ATPase gene from the budding yeast Saccharomyces cerevisiae (Saltzgaber, J., Kunapuli, S., and Douglas, M. G. (1983) J. Biol. Chem. 258, 11465-11470). The mRNAs which hybridized to pMa1 and pMa2 were translated by a reticulocyte lysate into polypeptides of Mr = 59,000 and 54,000, respectively. These genes products reacted with an anti-F1-ATPase serum and therefore correspond most probably to precursors of the alpha and beta subunits.     

Assembly of the mitochondrial membrane system. Characterization of COR1, the structural gene for the 44-kilodalton core protein of yeast coenzyme QH2-cytochrome c reductase

The respiratory deficiency of yeast strains previously assigned to complementation group G7 has been ascribed to the absence in the mutants of functional cytochrome b. Since G7 mutants are capable of synthesizing the apoprotein, the primary effect of the mutations is to prevent maturation of this electron carrier. The recombinant plasmid pG7/T1 with a 6.7-kilobase pairs (kb) insert of wild type yeast nuclear DNA has been selected from a genomic library by transformation of a G7 mutant to respiratory competency. The genetically active region of the pG7/T1 insert has been subcloned on a 3-kb fragment of DNA which has been shown to contain an open reading frame encoding a protein of 50,236 Mr. In situ disruption of the reading frame causes a deficiency in cytochrome b. The strain with the disrupted gene fails to complement G7 mutants thereby confirming the correct identification of the gene henceforth referred to as COR1. The carboxyl-terminal half of the COR1 gene has been fused to the amino-terminal half of the Escherichia coli trpE gene in the high expression vector pATH2. This plasmid construct promotes a high level of expression of the trpE/COR1 hybrid protein. Antibodies against the purified hybrid protein react with a 44 kDa protein subunit of yeast coenzyme QH2-cytochrome c reductase corresponding to the largest core subunit of the complex. These data indicate that the yeast nuclear gene COR1 codes for the 44-kDa core protein and that the latter is required for the conversion of apocytochrome b to mature cytochrome b.     

Mitochondrial translational-initiation and elongation factors in Saccharomyces cerevisiae

C155 and E252 are respiratory-defective mutants of Saccharomyces cerevisiae, previously assigned to complementation groups G37 and G142, respectively. The following evidence suggested that both mutants were likely to have lesions in components of the mitochondrial translational machinery: C155 and E252 display a pleiotropic deficiency in cytochromes a, a3 and b; both strains are severly limited in their ability to incorporate radioactive methionine into the mitochondrial translation products and, in addition, display a tendency to loose wild-type mitochondrial DNA. This set of characteristics is commonly found in strains affected in mitochondrial protein synthesis. To identify the biochemical lesions, each mutant was transformed with a wild-type yeast genomic library and clones complemented for the respiratory defect were selected for growth on a non-fermentable substrate. Analysis of the cloned genes revealed that C155 has a mutation in a protein which has high sequence similarity to bacterial elongation factor G and that E252 has a mutation in a protein homologous to bacterial initiation factor 2. Disruption of the chromosomal copy of each gene in a wild-type haploid yeast induced a phenotype analogous to that of the original mutants, but does not affect cell viability. These results indicate that both gene products function exclusively in mitochondrial protein synthesis. Subcloning of the IFM1 gene, coding for the mitochondrial initiation factor, indicates that the amino-terminal 423 residues of the protein are sufficient to promote peptide-chain initiation in vivo.     

Structure and evolution of a group of related aminoacyl-tRNA synthetases

A yeast nuclear gene, designated MSK1, has been selected from a yeast genomic library by transformation of a respiratory deficient mutant impaired in acylation of mitochondrial lysine tRNA. This gene confers a respiratory competent phenotype and restores the mutant's ability to acylate the mitochondrial lysine tRNA. The amino acid sequence of the protein encoded by MSK1 is homologous to yeast cytoplasmic lysyl-tRNA synthetase and to the product of the herC gene, which has recently been suggested to code for the Escherichia coli enzyme. These observations indicate that MSK1 codes for the lysyl-tRNA synthetase of yeast mitochondria. Several regions of high primary sequence conservation have been identified in the bacterial and yeast lysyl-tRNA synthetases. These domains are also present in the aspartyl- and asparaginyl-tRNA synthetases, further confirming the notion that all three present-day enzymes originated from a common ancestral gene. The most conserved domain, located near the carboxyl terminal ends of this group of synthetases is characterized by a cluster of glycines and is also highly homologous to the carboxyl-terminal region of the E. coli ammonia-dependent asparagine synthetase. A catalytic function of the carboxyl terminal domain is indicated by in vitro mutagenesis of the yeast mitochondrial lysyl-tRNA synthetase. Replacement of any one of three glycine residues by alanine and in one case by aspartic acid completely suppresses the activity of the enzymes, as evidenced by the inability of the mutant genes to complement an msk1 mutant, even when present in high copy. Other mutations result in partial loss of activity. Only one glycine replacement affects the stability of the protein in vivo. The observed presence of a homologous domain in asparagine synthetase, which, like the aminoacyl-tRNA synthetases, catalyzes the formation of an aminoacyladenylate, suggests that the glycine-rich sequence is part of a catalytic site involved in binding of ATP and of the aminoacyladenylate intermediate.     

Phenylalanyl-tRNA synthetase of wheat embryos. Purification, molecular weight, structure, properties]

From wheat germ, a phenylalanyl-tRNA synthetase (E.C.6.1.1.20) has been isolated and purified 187 fold by means of ammonium sulfate fractionation (40-50 per cent) followed by Sephadex G-200 gel filtration, chromatographies on DEAE-cellulose and hydroxyapatite. The enzyme appears to be homogeneous on Sephadex G-200 molecular filtration and polyacrylamide gel electrophoresis. Molecular weight determinations by sucrose gradient centrifugation, gel filtration and gel electrophoresis give an average of 250 00 daltons. The enzyme is dissociated in 1 per cent sodium dodecyl sulfate into two different equimolar components of 80 000 and 50 000 daltons ; this result suggests that the phenylalanyl-tRNA synthetase has a subunit structure : alpha2 beta2. Dissociation with sodium dodecyl sulfate and dithiothreitol gives four other components, probably resulting from the breakdown of the subunits. Optima values of pH, Mg2+ and K+ concentrations, effect of SH-compnents, kinetic parameters have been determined in the aminoacylation reaction. Physical and catalytic properties of wheat germ phenylalanyl-tRNA synthetase appear very similar to those of the yeast and E. coli enzymes.     

Cloning and characterization of the gene for the yeast cytoplasmic threonyl-tRNA synthetase.

A fragment of DNA from the yeast nuclear gene MST1 that codes for the mitochondrial tRNAThr1 synthetase was used as a probe to screen for other yeast threonyl-tRNA synthetase genes. At low stringency, the MST1 probe hybridizes strongly to a 6.6 kb EcoRI fragment of yeast genomic DNA with the homologous gene and in addition hybridizes more weakly to a smaller 3.6 kb EcoRI fragment with a second threonyl-tRNA synthetase gene (THS1). To clone THS1, a library was constructed by ligation to pUC18 of size selected (3-4.5 kb) EcoRI fragments of genomic DNA. Several clones containing the 3.6 kb EcoRI fragment were isolated. A 2,202 nucleotide long open reading frame corresponding to THS1 has been identified in the cloned fragment of DNA. The predicted protein encoded by THS1 is 38% identical to the E. coli threonyl-tRNA synthetase over the latter's length (642 amino acids) and is 42% identical to the predicted MST1 product over its 462 residues. In situ disruption of the chromosomal copy of THS1 is lethal to the cell, indicating that this gene codes for the cytoplasmic threonyl-tRNA synthetase.     

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.     

Identification of the pheS5 mutation, which causes thermosensitivity of Escherichia coli mutant NP37.

The pheS5 mutation responsible for the thermosensitive phenylalanyl-tRNA synthetase of the classical Escherichia coli NP37 was cloned by a recombination event and identified by DNA sequence analysis. The mutation was subsequently verified by direct sequencing of amplified NP37 DNA generated by an asymmetric polymerase chain reaction. The resulting amino acid exchange, Gly-98 to Asp-98 in the phenylalanyl-tRNA synthetase alpha subunit, might cause subunit disaggregation due to electrostatic repulsion.     

Molecular cloning and nucleotide sequence of the gene for Escherichia coli leucyl-tRNA synthetase.

The gene for Escherichia coli leucyl-tRNA synthetase leuS has been cloned by complementation of a leuS temperature sensitive mutant KL231 with an E.coli gene bank DNA. The resulting clones overexpress leucyl-tRNA synthetase (LeuRS) by a factor greater than 50. The DNA sequence of the complete coding regions was determined. The derived N-terminal protein sequence of LeuRS was confirmed by independent protein sequencing of the first 8 aminoacids. Sequence comparison of the LeuRS sequence with all aminoacyl-tRNA synthetase sequences available reveal a significant homology with the valyl-, isoleucyl- and methionyl-enzyme indicating that the genes of these enzymes could have derived from a common ancestor. Sequence comparison with the gene product of the yeast nuclear NAM2-1 suppressor allele curing mitochondrial RNA maturation deficiency reveals about 30% homology.     

Interaction of the tRNA(Phe) 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].     

Mechanism of discrimination between cognate and non-cognate tRNAs by phenylalanyl-tRNA synthetase from yeast.

The interaction between phenylalanyl-tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNA1Val (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titrations and fast kinetic techniques. The fluorescence of the Y-base of tRNAPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators. 1. Specific complexes between phenylalanyl-tRNA synthetase and tRNAPhe from yeast are formed in a two-step mechanism: a nearly diffusion-controlled recombination is followed by a fast conformational transition. Binding constants, rate constants and changes in the quantum yield of the Y-base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt, concentration of Mg2+ ions and temperature. 2. Heterologous complexes between phenylalanyl-tRNA synthetase (E. coli) and tRNAPhe (yeast) are formed in a similar two-step mechanism as the specific complexes; the conformational transition, however, is slower by a factor 4-5. 3. Formation of non-specific complexes between phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) proceeds in a one-step mechanism. Phenylalanyl-tRNA synthetase (yeast) binds either two molecules of tRNAPhe (yeast) or only one molecule of tRNATyr (E. coli); tRNA1Val (E. coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) are determined under a variety of conditions. In contrast to specific complex formation, non-specific binding is disfavoured by the presence of Mg2+ ions, and is not affected by pH and the presence of pyrophosphate. The difference in the stabilities of specific and non-specific complexes can be varied by a factor of 2--100 depending on the ionic conditions. Discrimination of cognate and non-cognate tRNA by phenylalanyl-tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions.     

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).     

Identification of clones carrying an E. coli tRNAPhe gene by suppression of phenylalanyl-tRNA synthetase thermosensitive mutants

Two libraries of cloned E. coli DNA were screened for plasmids which complemented thermosensitive phenylalanyl-tRNA synthetase mutants. Four plasmids were isolated which complemented pheS and pheT thermosensitive mutations but which do not carry pheS or pheT, the structural genes for phenylalanyl-tRNA synthetase. All these plasmids increased the intracellular tRNAPhe concentration. Three plasmids were shown to carry the structural gene for tRNAPhe which we call pheU. By restriction enzyme analysis, DNA blotting and DNA:tRNA hybridization, pheU was localised to a 280 bp fragment within a 5.6 kb PstI restriction fragment of E.coli DNA.     

Nuclear genes encoding the yeast mitochondrial ATPase complex. Analysis of ATP1 coding the F1-ATPase alpha-subunit and its assembly

Mitochondria prepared from the yeast nuclear pet mutant N9-84 lack a detectable F1-ATPase activity. Genetic complementation of this mutant with a pool of yeast genomic DNA in the yeast Escherichia coli shuttle vector YEp13 restored its growth on a nonfermentable carbon source. Mitochondria prepared from the transformed host contained an 8-fold higher than normal level of the F1 alpha-subunit and restored ATPase activity to 50% that of the wild-type strain. Deletion and nucleotide sequence analysis of the complementing DNA on the plasmid revealed a coding sequence designated ATP1 for a protein of 544 amino acids which exhibits 60 and 54% direct protein sequence homology with the proton-translocating ATPase alpha-subunits from tobacco chloroplast and E. coli, respectively. In vitro expression and mitochondrial import experiments using this ATP1 sequence showed that additional amino-terminal sequences not present in the comparable plant and bacterial subunits function as transient sequences for import.