methionyl-tRNA Transformylase Research Articles

A Disease-causing Point Mutation in Human Mitochondrial tRNAMet Results in tRNA Misfolding Leading to Defects in Translational Initiation and Elongation

The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNA(Met) (hmtRNA(Met)) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNA(Met) with a C8. The hmtRNA(Met) serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U-->C mutation leads to a failure of the tRNA to respond conformationally to Mg(2+). This mutation results in a drastic disruption of the structure of the hmtRNA(Met), which significantly reduces its aminoacylation. The small fraction of hmtRNA(Met) that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg(2+), the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg(2+), the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg(2+)-binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans.

5-Formyltetrahydrofolate Is an Inhibitory but Well Tolerated Metabolite in Arabidopsis Leaves

5-Formyltetrahydrofolate (5-CHO-THF) is formed via a second catalytic activity of serine hydroxymethyltransferase (SHMT) and strongly inhibits SHMT and other folate-dependent enzymes in vitro. The only enzyme known to metabolize 5-CHO-THF is 5-CHO-THF cycloligase (5-FCL), which catalyzes its conversion to 5,10-methenyltetrahydrofolate. Because 5-FCL is mitochondrial in plants and mitochondrial SHMT is central to photorespiration, we examined the impact of an insertional mutation in the Arabidopsis 5-FCL gene (At5g13050) under photorespiratory (30 and 370 micromol of CO2 mol(-1)) and non-photorespiratory (3200 micromol of CO2 mol(-1)) conditions. The mutation had only mild visible effects at 370 micromol of CO2 mol(-1), reducing growth rate by approximately 20% and delaying flowering by 1 week. However, the mutation doubled leaf 5-CHO-THF level under all conditions and, under photorespiratory conditions, quadrupled the pool of 10-formyl-/5,10-methenyltetrahydrofolates (which could not be distinguished analytically). At 370 micromol of CO2 mol(-1), the mitochondrial 5-CHO-THF pool was 8-fold larger in the mutant and contained most of the 5-CHO-THF in the leaf. In contrast, the buildup of 10-formyl-/5,10-methenyltetrahydrofolates was extramitochondrial. In photorespiratory conditions, leaf glycine levels were up to 46-fold higher in the mutant than in the wild type. Furthermore, when leaves were supplied with 5-CHO-THF, glycine accumulated in both wild type and mutant. These data establish that 5-CHO-THF can inhibit SHMT in vivo and thereby influence glycine pool size. However, the near-normal growth of the mutant shows that even exceptionally high 5-CHO-THF levels do not much affect fluxes through SHMT or any other folate-dependent reaction, i.e. that 5-CHO-THF is well tolerated in plants.

Transfer RNA Labeling of Escherichia coli Methionyl-tRNA Transformylase

Homogeneous methionyl-tRNA transformylase from Escherichia coli can react with periodate-treated tRNAMetf to form a Schiff's base through a free amino group (probably the epsilon-amino group of a lysine) and the 2',3'-aldehyde groups created at the 3'-terminal ribose of tRNA. This reaction is reflected by the loss of activity of the enzyme at saturating tRNA dialdehyde and upon reduction of the Schiff's base by NaBH4. the kinetics of inactivation (37 degrees C, pH 8.5) level off at 50% of the initial enzymic activity. In the presence of 2 mM cyanohydridoborate, a mild reducing agent which leaves intact the reacting aldehyde groups of oxidized tRNA but continuously reduces the Schiff's base in equilibrium, the activity of the enzyme can be destroyed by 100%, at a rate of 0.044 min-1, with the parallel covalent incorporation of close to one tRNA molecule per enzyme molecule. Selectivity of the labeling is also supported by the demonstration that, prior to Schiff's base formation, modified tRNA binds the transformylase with equilibrium constant and stoichiometry in good agreement with those for the active binding of unmodified tRNa. Moreover intact tRNA competes for the inactivation by the dialdehyde derivative with an affinity constant identical to that for its active binding to the enzyme

Recognition of tRNAs by Methionyl-tRNA Transformylase from Mammalian Mitochondria

Protein synthesis involves two methionine-isoaccepting tRNAs, an initiator and an elongator. In eubacteria, mitochondria, and chloroplasts, the addition of a formyl group gives its full functional identity to initiator Met-tRNA(Met). In Escherichia coli, it has been shown that the specific action of methionyl-tRNA transformylase on Met-tRNA(f)(Met) mainly involves a set of nucleotides in the acceptor stem, particularly a C(1)A(72) mismatch. In animal mitochondria, only one tRNA(Met) species has yet been described. It is admitted that this species can engage itself either in initiation or elongation of translation, depending on the presence or absence of a formyl group. In the present study, we searched for the identity elements of tRNA(Met) that govern its formylation by bovine mitochondrial transformylase. The main conclusion is that the mitochondrial formylase preferentially recognizes the methionyl moiety of its tRNA substrate. Moreover, the relatively small importance of the tRNA acceptor stem in the recognition process accounts for the protection against formylation of the mitochondrial tRNAs that share with tRNA(Met) an A(1)U(72) motif.

Expression and characterization of bovine mitochondrial methionyl-tRNA transformylase.

Translational initiation in bacteria and some organelles such as mitochondria and chloroplasts requires formyl-methionyl-tRNA (fMet-tRNA). Methionyl-tRNA (Met-tRNA) undergoes formylation by methionyl-tRNA transformylase (MTF), and the resulting fMet-tRNA is utilized exclusively in the initiation process. The gene encoding mammalian mitochondrial MTF (MTFmt) was cloned recently. When the cDNA corresponding to mature MTFmt was cloned into an expression vector, no expression of MTFmt was observed. However, if the cDNA was fused with the histidine-tag sequence at the N-terminus, MTFmt could be expressed in Escherichia coli. The recombinant enzyme was purified by a single step on a histidine-binding metal affinity column. We previously found that native MTFmt is able to formylate E. coli elongator Met-tRNA as well as the initiator Met-tRNA. The specific formylation of the initiator Met-tRNA by E. coli MTF is quite important in bacterial translational initiation. The purified recombinant MTFmt with the histidine-tag showed almost identical kinetic parameters to those of native MTFmt. This expression system is suitable for the rapid, efficient production of MTFmt in amounts adequate for further biophysical studies, which will provide another approach for elucidating the formylation mechanism, in addition to studies on E. coli MTF.

Mammalian Mitochondrial Methionyl-tRNA Transformylase from Bovine Liver: PURIFICATION, CHARACTERIZATION, AND GENE STRUCTURE

The mammalian mitochondrial methionyl-tRNA transformylase (MTFmt) was partially purified 2,200-fold from bovine liver mitochondria using column chromatography. The polypeptide responsible for MTFmt activity was excised from a sodium dodecyl sulfate-polyacrylamide gel and the amino acid sequences of several peptides were determined. The cDNA encoding bovine MTFmt was obtained and its nucleotide sequence was determined. The deduced amino acid sequence of the mature form of MTFmt consists of 357 amino acid residues. This sequence is about 30% identical to the corresponding Escherichia coli and yeast mitochondrial MTFs. Kinetic parameters governing the formylation of various tRNAs were obtained. Bovine MTFmt formylates its homologous mitochondrial methionyl-tRNA and the E. coli initiator methionyl-tRNA (Met-tRNAfMet) with essentially equal efficiency. The E. coli elongator methionyl-tRNA (Met-tRNAmMet) was also formylated although with somewhat less favorable kinetics. These results suggest that the substrate specificity of MTFmt is not as rigid as that of the E. coli MTF which clearly discriminates between the bacterial initiator and elongator Met-tRNAs. These observations are discussed in terms of the presence of a single tRNAMet gene in mammalian mitochondria.

Mammalian Mitochondrial Methionyl-tRNA Transformylase from Bovine Liver: PURIFICATION, CHARACTERIZATION, AND GENE STRUCTURE

Mammalian Mitochondrial Methionyl-tRNA Transformylase from Bovine Liver: PURIFICATION, CHARACTERIZATION, AND GENE STRUCTURE

Important Role of the Amino Acid Attached to tRNA in Formylation and in Initiation of Protein Synthesis in Escherichia coli

In attempts to convert an elongator tRNA to an initiator tRNA, we previously generated a mutant elongator methionine tRNA carrying an anticodon sequence change from CAU to CUA along with the two features important for activity of Escherichia coli initiator tRNA in initiation. This mutant tRNA (Mi:2 tRNA) was active in initiation in vivo but only when aminoacylated with methionine by overproduction of methionyl-tRNA synthetase. Here we show that the Mi:2 tRNA is normally aminoacylated in vivo with lysine and that the tRNA aminoacylated with lysine is a very poor substrate for formylation compared with the same tRNA aminoacylated with methionine. By introducing further changes at base pairs 4:69 and 5:68 in the acceptor stem of the Mi:2 tRNA to those found in the E. coli initiator tRNA, we show that change of the U4:A69 base pair to G4:C69 and overproduction of lysyl-tRNA synthetase and methionyl-tRNA transformylase results in partial formylation of the mutant tRNA and activity of the formyllysyl-tRNAs in initiation of protein synthesis. Thus, the G4: C69 base pair contributes toward formylation of the tRNA and protein synthesis in E. coli can be initiated with formyllysine. We also discuss the implications of these and other results on recognition of tRNAs by E. coli lysyl-tRNA synthetase and on competition in cells among aminoacyl-tRNA synthetases.

Escherichia coli initiator tRNA: structure-function relationships and interactions with the translational machinery.

We showed previously that the sequence and (or) structural elements important for specifying the many distinctive properties of Escherichia coli initiator tRNA are clustered in the acceptor stem and in the anticodon stem and loop. This paper briefly describes this and reviews the results of some recently published studies on the mutant initiator tRNAs generated during this work. First, we have studied the effect of overproduction of methionyl-tRNA transformylase (MTF) and initiation factors IF2 and IF3 on activity of mutant initiator tRNAs that are defective at specific steps in the initiation pathway. Overproduction of MTF rescued specifically the activity of mutant tRNAs defective in formylation but not mutants defective in binding to the P site. Overproduction of IF2 increased the activity of all mutant tRNAs having the CUA anticodon but not of mutant tRNA having the GAC anticodon. Overproduction of IF3 had no effect on the activity of any of the mutant tRNAs tested. Second, for functional studies of mutant initiator tRNA in vivo, we used a CAU --> CUA anticodon sequence mutant that can initiate protein synthesis from UAG instead of AUG. In contrast with the wild-type initiator tRNA, the mutant initiator tRNA has a 2-methylthio-N6-isopentenyl adenosine (ms2i6A) base modification next to the anticodon. Interestingly, this base modification is now important for activity of the mutant tRNA in initiation. In a miaA strain of E. coli deficient in biosynthesis of ms2i6A, the mutant initiator tRNA is much less active in initiation. The defect is specifically in binding to the ribosomal P site.

Heteronuclear NMR studies of the interactions of 15N-labeled methionine-specific transfer RNAs with methionyl-tRNA transformylase

In Escherichia coli the methionylated initiator methionyl-tRNA (tRNAfMet) is formylated on the aminoacyl moiety by the enzyme methionyl-tRNA transformylase. The methionylated elongator methionyl-tRNA (tRNAmMet) is not modified in this way. In order to gain structural information about this specific recognition, solution NMR studies were carried out. To be able to identify changes that were occurring in the tRNA molecule on interaction with the methionyl-tRNA transformylase, the imino protons involved in secondary and tertiary base pairing in the tRNAfMet and tRNAmMet molecules first had to be assigned to specific resonances in the NMR spectra. A combination of 2D NOESY, 2D HMQC, and 3D NOESY--HMQC spectra were used on uniformly 15N-labeled samples. After assignment of the base pairs of the tRNA, the two forms of tRNA were separately mixed with transformylase in a 1:1 molar ratio. The HMQC spectra of both the tRNAmMet and the tRNAfMet showed general broadening, but in the tRNAfMet HMQC spectra a decrease in the intensity of several resonances was also observed. These resonances had been assigned to the acceptor stem of the tRNA, confirming site-directed mutagenesis experiments that it is the acceptor stem of the tRNA which is important in conferring the specificity for the transformylase. The loss of intensity of the acceptor stem resonances suggests that this part of tRNAfMet melts upon binding to the enzyme.

Mutants of Escherichia coli Initiator tRNA Defective in Initiation: EFFECTS OF OVERPRODUCTION OF METHIONYL-tRNA TRANSFORMYLASE AND THE INITIATION FACTORS IF2 AND IF3

We describe the effects of overproduction of methionyl-tRNA transformylase and initiation factors IF2 and IF3 on the activity, in vivo, of initiator tRNA mutants defective at specific steps of the initiation process in protein synthesis. The activity of the U35A36/G72 and U35A36/G72G73 mutants, which are defective in formylation, was increased by overproduction of methionyl-tRNA transformylase. In contrast, the activity of the C30:G40/U35A36 mutant, which is formylated normally but is defective in binding to the ribosomal P site, was not increased. Overproduction of IF2 had a strong stimulatory effect on the activity of virtually all the mutants carrying the U35A36 anticodon sequence change, including the U35A36, U35A36/G72, U35A36/G72G73, and the C30:G40/U35A36 mutants. In cells overproducing IF2, the amount of protein made by translation of a mutant mRNA, which uses the U35A36 mutant initiator tRNA, is severalfold higher than that made by translation of a wild type mRNA. We discuss the possible implications of this result on overproduction of proteins and on the order of assembly of the 30 S ribosome.mRNA.fMet-tRNA initiation complex in Escherichia coli. Over-production of IF3 did not affect the initiator activity of any of the tRNA mutants studied.

Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation.

Deformylase performs an essential step in the maturation of proteins in eubacteria, by removing the formyl group from the N-terminal methionine residue of ribosome-synthesized polypeptides. In spite of this important role in translation, the enzyme had so far eluded characterization because of its instability. We report the isolation of the deformylase gene of Escherichia coli, def, by overexpression of a genomic library from a high-copy-number plasmid and selection for utilization of the substrate analogue formyl-leucyl-methionine as a source of methionine. The def gene encodes a 169 amino acid polypeptide that bears no obvious resemblance to other known proteins. It forms an operon with the fmt gene, that encodes the initiator methionyl-tRNA(i) transformylase, which was recently characterized (Guillon et al., J. Bacteriol., 174, 4294-4301, 1992). This operon was mapped at min 72 of the E. coli chromosome. The def gene could be inactivated if the fmt gene was also inactivated, or if biosynthesis of N10-formyl-tetrahydrofolate, the formyl donor in methionyl-tRNA(i) transformylation, was blocked by trimethoprim. These findings designate deformylase as a target for antibacterial chemotherapy.

Relationship between the structure and function of Escherichia coli initiator tRNA

Through functional studies of mutant tRNAs, we have identified sequence and/or structural features important for specifying the many distinctive properties of E coli initiator tRNA. Many of the mutant tRNAs contain an anticodon sequence change from CAU→CUA and are now substrates for E coli glutaminyl-tRNA synthetase (GlnRS). We describe here the effect of further mutating the discriminator base 73 and nucleotide 72 at the end of the acceptor stem on: i) recognition of the mutant tRNAs by E coli GlnRS; ii) recognition by E coli methionyl-tRNA transformylase; and iii) activity of the mutant tRNAs in initiation in E coli. For GlnRS recognition, our results are, in general, consistent with interactions found in the crystal structure of the E coli GlnRS-glutamine tRNA complex. The results also support our previous conclusion that formylation of initiator tRNA is important for its function in initiation.

Structural and sequence elements important for recognition of Escherichia coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem.

We show that the structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. This conclusion is based on mutagenesis of the E. coli initiator tRNA gene followed by measurement of kinetic parameters for formylation of the mutant tRNAs in vitro and function in protein synthesis in vivo. The first base pair found at the end of the amino acid acceptor stem in all other tRNAs is replaced by a C.A. "mismatch" in E. coli initiator tRNA. Mutation of this C.A. to U:A, a weak base pair, or U.G., a mismatch, has little effect on formylation, whereas mutation to C:G, a strong base pair, has a dramatic effect lowering Vmax/Kappm by 495-fold. Mutation of the second basepair G2:C71 to U2:A71 lowers Vmax/Kappm by 236-fold. Replacement of the third base-pair C3:G70 by U3:A70, A3:U70, or G3:C70 lowers Vmax/Kappm by about 67-, 27-, and 30-fold, respectively. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and T psi C stem have little or no effect on formylation.

Mitochondrial folates and methionyl-tRNA transformylase activity during germination and early growth of seeds

Mitochondria were isolated from the cotyledons of pea ( Pisum sativum cv Homesteader) and peanut ( Arachis hypogaea cv Early Spanish) seeds over a 7-day growth period. The rate of mitochondrial oxygen uptake increased 3-4-fold during the first 4 days of growth and parallel changes were observed in the respiratory control and ADP/O ratios. In both species, the total cotyledonary pool of folate derivatives increased 3-4-fold during this period of germination whereas that associated with isolated mitochondria increased 5-10-fold. Until day 3 of growth, the mitochondrial folates were principally polyglutamates of 10-formyltetrahydrofolate but between day 4 and day 7 increasing levels of 5-methyltetrahydrofolate polyglutamates were detected. Pea and peanut mitochondria contained methionyl-tRNA transformylase (EC 2.1.2.9) activity that displayed an absolute requirement for 10-formyl-tetrahydrofolate. The specific activity of this enzyme rose during germination, reaching maximal levels between days 3 and 4. Isolated pea mitochondria had the ability to incorporate [ 3H]leucine and [ 35S]methionine into protein in a reaction that required ADP and malate but was strongly inhibited by chloramphenicol. Organelles isolated after 4 days of germination incorporated leucine at rates ca 5-fold greater than shown by mitochondria of 16-hour-old seedlings. The inter-relationships between respiratory activity, mitochondrial formyltetrahydrofolates and methionyl-tRNA transformylase activity suggest a role for organelle protein synthesis during germination of these legume species.

Inhibitor studies of methionyl-tRNA transformylase of Euglena gracilis

Inhibitor studies of the only known eukaryotic methionyl-tRNA transformylase (10-formyltetrahydrofolate: l-methionyl-tRNA N-transformylase, EC 2.1.2.9) were carried out. All the natural pteroylglutamic acid derivatives examined, with the exception of pteroylglutamic acid, are inhibitors. The most effective is 5-methyltetrahydrofolate (5-CH 3-H 4PteGlu) ( K I = 3 · 10 −6 M), which is the only noncompetitive inhibitor of the enzyme. All the other derivatives tested are competitive, and H 4PteGlu shows a cooperative inhibition. These and other data obtained with pteroylglutamic analogues show that, in contrast to the bacterial enzyme, Euglena transformylase is also inhibited by compounds without a fully reduced pyrazine ring and is very sensitive to compounds with a methyl group in position 5 or 10 of the pteridine ring.

Methionyl-transfer-RNA transformylase from Escherichia coli. Purification and characterisation.

A large‐scale purification procedure of methionyl‐tRNA transformylase (10‐formyltetrahydro‐folate:l‐methionyl‐tRNA N‐formyltransferase) from Escherichia coli is described. The purification (10000‐fold) produces 22 mg homogeneous enzyme from 5 kg wet cells with a yield of 45%. The specific activity of the enzyme in the reaction of formylation of methionyl‐tRNAMetf is 30‐fold higher than that reported by others [Dickerman, H. W., Steers, E., Jr, Redfield, B. G. and Weissbach, H. (1967) Biol. Chem. 242, 1522‐1525].The enzyme is a monomer of molecular weight 32000, based on high‐speed equilibrium sedimentation, small‐angle neutron scattering, molecular sieving on Sephadex and gel electrophoresis in the presence of sodium dodecyl sulfate. Its absorption coefficient has been determined as A280nm=1.4 ± 0. 1 cm2. mg−1.The Michaelian parameters of the transformylation reaction have been measured for each substrate at 25 °C, pH 7. 6, in the presence of 150 mM KC1 and 7 mM MgCl2. Km values are 13.5 ± 1.5 μ and 0.35 ± 0.05 μ for 10‐formyltetrahydrofolate and for methionyl‐tRNAMetf respectively. A V value of 20 ± 2 s−1 is found at saturation of both substrates.The tryptophan fluorescence of transformylase is remarkably sensitive to the bindings of 10‐formyltetrahydrofolate and tRNAMetf This property enables one to determine 1:1 stoichiometries of the enzyme with either 10‐formyltetrahydrofolate, tRNAMetf at high ionic strength. Stability of the tRNAMetf transformylase complex is improved upon aminoacylation of tRNA. The observed association constant for the interaction between enzyme and tRNAMetf is sensitive to the concentration of cations (K+ or/and Mg2+). This behaviour has allowed us to estimate to 4.6 ± 0.7 as the number of ion pairs formed between tRNAMetf and transformylase.

The primary structure of non-initiator methionine transfer ribonucleic acid from Bakers' yeast. I. Purification and complete digestion with ribonuclease T1 and pancreatic ribonuclease A.

The methionine acceptor activity of a crude tRNA from bakers' yeast was resolved into two peaks (I and II) by column chromatography on DEAE-Sephadex A-25 with a 1 M phosphate system. Methionine tRNA from peak II was not formylated by E. coli methionyl-tRNA transformylase [EC 2.1.2.9.] after being charged with methionine, whereas that from peak I was formylatable under the same conditions. A substantial amount of unlabelled methionine tRNA, tRNAMetm, was highly purified from the peak II fraction by successive chromatographic procedures. The purified tRNAMetm was digested with pancreatic ribonuclease A [EC 3.1.4.22] and ribonuclease T1 [EC 3.1.4.8]. The digestion products were isolated into individual components and completely sequenced. The results of sequence analysis of the two RNase digests were in good agreement and indicated that the chain length of this tRNA is 76, including 13 modified nucleotides. These oligonucleotide fragments can be constructed into a unique total sequence, assuming a few conventional features of clover leaf structure for the tRNA was established by analyses of partial digestion products with RNase T1, as reported in the accompanying paper.

Increased mitochondrial leucyl- and phenylalanyl-tRNA synthetase activity as a result of inhibition of mitochondrial protein synthesis.

THE synthesis of certain mitochondrial proteins is inhibited by cycloheximide, an inhibitor of cytoplasmic protein synthesis, and not by the inhibitors of mitochondrial protein synthesis, chloramphenicol and ethidium bromide1,2. This suggests that these proteins are synthesised on cytoplasmic ribosomes from messenger RNA (mRNA) transcribed in the nucleus. Their formation, however, is dependent, in some way, on mitochondrial protein synthesis, as growth in the presence of inhibiting concentrations of either ethidium bromide or chloramphenicol results in great increases in the specific activity of mitochondrial DNA-dependent RNA polymerase1, elongation factors, and methionyl-tRNA transformylase, as well as an increase in mitochondrial ribosomal proteins2. This led Barath and Kuntzel1 to propose that the increased synthesis of mitochondrial proteins results from a disruption of mitochondrial control over transcription of nuclear genes specifying the structure of mitochondrial proteins. The model proposes a represser protein, specified and produced by the mitochondrion, that regulates transcription of mitochondrion-specific genes in the nucleus. When synthesis of the putative represser is inhibited by either ethidium bromide or chloramphenicol, the target nuclear genes are transcribed at an increased rate and the production of mitochondrial proteins increases. Although a direct test of Barath and Kuntzel's model would involve identifying the repressor and its targets, some of the underlying assumptions may be checked by measuring the production by Neurospora of the mitochondrial leucyl-tRNA synthetase, whose structure is known to be determined by a nuclear gene in the Leu-5 region of linkage group V (refs 3 and 4, and our unpublished results). In this case the site of mRNA synthesis is deduced from formal genetic analysis rather than from patterns of inhibition by inhibitors of protein synthesis. As an internal control we also followed the synthesis of the mitochondrial phenylalanyl-tRNA synthetase (whose genetics is unknown). We found that the synthesis of the leucyl-tRNA synthetase in response to chloramphenicol, ethidium bromide and cycloheximide fully conformed to Barath and Kuntzel's model. The production of phenylalanyl-tRNA synthetase activity in response to the inhibitors, however, suggests the involvement of either activation of extant enzyme or processing of an inactive precursor molecule as a large component of the increase in the specific activity of the enzyme after treatment with chloramphenicol.

Variations in activity of aminoacyl-tRNA synthetases as a function of development in Bacillus subtilis

Extracts from Bacillus sublilis cells at various stages of growth and spores were assayed for aminoacyl-tRNA synthetase and methionyl-tRNA transformylase activity. There was no major change in any synthetase activity or in methionyl-tRNA transformylase activity during the sporulation cycle, which implies that these are not sporulation induced enzymes. However, extracts from B. subtilis cultures showed a burst of activity of aminoacyl-tRNA synthetases during exponential growth. Preparations from dormant spores possessed the same kinds of aminoacyl-tRNA synthetase activities as vegetative cells for all the amino acids which were studied. Spores also contained methionyl-tRNA transformylases. These findings suggest that spores ought to be able to aminoacylate tRNA and formylate the initiator. N-formylmethionyl-tRNA, immediately upon germination.