The Mere Lack of rT Modification in Initiator tRNA Does Not Facilitate Formylation-Independent Initiation in Escherichia coli

Formylation of initiator methionyl-tRNA is essential for normal growth of eubacteria. However, under special conditions, it has been possible to initiate protein synthesis with unformylated initiator tRNA even in eubacteria. Earlier studies suggested that the lack of ribothymidine (rT) modification in initiator tRNA may facilitate initiation in the absence of formylation. In this report we show, by using trmA strains of Escherichia coli (defective for rT modification) and a sensitive in vivo initiation assay system, that the lack of rT modification in the initiators is not sufficient to effect formylation-independent initiation of protein synthesis.     

Role of methionine and formylation of initiator tRNA in initiation of protein synthesis in Escherichia coli.

We showed recently that a mutant of Escherichia coli initiator tRNA with a CAU-->CUA anticodon sequence change can initiate protein synthesis from UAG by using formylglutamine instead of formylmethionine. We further showed that coupling of the anticodon sequence change to mutations in the acceptor stem that reduced Vmax/Km(app) in formylation of the tRNAs in vitro significantly reduced their activity in initiation in vivo. In this work, we have screened an E. coli genomic DNA library in a multicopy vector carrying one of the mutant tRNA genes and have found that the gene for E. coli methionyl-tRNA synthetase (MetRS) rescues, partially, the initiation defect of the mutant tRNA. For other mutant tRNAs, we have examined the effect of overproduction of MetRS on their activities in initiation and their aminoacylation and formylation in vivo. Some but not all of the tRNA mutants can be rescued. Those that cannot be rescued are extremely poor substrates for MetRS or the formylating enzyme. Overproduction of MetRS also significantly increases the initiation activity of a tRNA mutant which can otherwise be aminoacylated with glutamine and fully formylated in vivo. We interpret these results as follows. (i) Mutant initiator tRNAs that are poor substrates for MetRS are aminoacylated in part with methionine when MetRS is overproduced. (ii) Mutant tRNAs aminoacylated with methionine are better substrates for the formylating enzyme in vivo than mutant tRNAs aminoacylated with glutamine. (iii) Mutant tRNAs carrying formylmethionine are significantly more active in initiation than those carrying formylglutamine. Consequently, a subset of mutant tRNAs which are defective in formylation and therefore inactive in initiation when they are aminoacylated with glutamine become partially active when MetRS is overproduced.     

Suppressor mutations in Escherichia coli methionyl-tRNA formyltransferase that compensate for the formylation defect of a mutant tRNA aminoacylated with lysine

The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in eubacteria such as Escherichia coli. In addition to the determinants for formylation present in the initiator tRNA, the nature of the amino acid attached to the tRNA is also important for formylation. We showed previously that a mutant tRNA aminoacylated with lysine was an extremely poor substrate for formylation. As a consequence, it was essentially inactive in initiation of protein synthesis in E. coli. In contrast, the same tRNA, when aminoacylated with methionine, was a good substrate for formylation and was, consequently, quite active in initiation. Here, we report on the isolation of suppressor mutations in MTF which compensate for the formylation defect of the mutant tRNA aminoacylated with lysine. The suppressor mutant has glycine 178 changed to glutamic acid. Mutants with glycine 178 of MTF changed to aspartic acid, lysine, and leucine were generated and were found to be progressively weaker suppressors. Studies on allele specificity of suppression using different mutant tRNAs as substrates suggest that the Gly178 to Glu mutation compensates for the nature of the amino acid attached to the tRNA. We discuss these results in the framework of the crystal structure of the MTF.fMet-tRNA complex published recently.     

Expression of Escherichia coli methionyl-tRNA formyltransferase in Saccharomyces cerevisiae leads to formylation of the cytoplasmic initiator tRNA and possibly to initiation of protein synthesis with formylmethionine

Protein synthesis in eukaryotic cytoplasm and in archaebacteria is initiated with methionine, whereas, that in eubacteria and in eukaryotic organelles, such as mitochondria and chloroplasts, is initiated with formylmethionine. In view of this clear distinction, we have investigated whether protein synthesis in the eukaryotic cytoplasm can be initiated with formylmethionine, and, if so, what the consequences are to the cell. For this purpose, we have expressed in an inducible manner the Escherichia coli methionyl-tRNA formyltransferase (MTF) in the cytoplasm of the yeast Saccharomyces cerevisiae. Expression of active MTF, but not of an inactive mutant, leads to formylation of methionine attached to the yeast cytoplasmic initiator tRNA to the extent of about 70%. As a consequence, the yeast strain grows slowly. Coexpression of the E. coli polypeptide deformylase (DEF), which removes the formyl group from the N-terminal formylmethionine in a polypeptide, rescues the slow-growth phenotype, whereas, coexpression of an inactive mutant of DEF does not. These results suggest that the cytoplasmic protein-synthesizing system of yeast, like that of eubacteria, can at least to some extent utilize formylated initiator Met-tRNA to initiate protein synthesis and that initiation of proteins with formylmethionine leads to the slow-growth phenotype. Removal of the formyl group in these proteins by DEF would explain the rescue of the slow-growth phenotype.     

Isolation and partial characterization of three Escherichia coli mutants with altered transfer ribonucleic acid methylases.

Seven transfer ribonucleic acid (tRNA) methylase mutants were isolated from Escherichia coli K-12 by examining the ability of RNA prepared from clones of unselected mutagenized cells to accept methyl groups from S-adenosylmethionine catalyzed by crude enzymes from wild-type cells. Five of the mutants had an altered uracil-tRNA methylase; consequently their tRNA's lacked ribothymidine. One mutant had tRNA deficient in 7-methylguanosine, and one mutant contained tRNA lacking 2-thio-5-methylaminomethyluridine. The genetic loci of the three tRNA methylase mutants were distributed over the E. coli genome. The mutant strain deficient in 7-methylguanosine biosynthesis showed a reduced efficiency in the suppression of amber mutations carried by T4 or lambda phages.     

Initiation of protein synthesis in Saccharomyces cerevisiae mitochondria without formylation of the initiator tRNA

Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNA(fMet)) for initiation. Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNA(fMet)). The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and uses N(10)-formyltetrahydrofolate (10-formyl-THF) as the formyl donor. We have studied yeast mutants carrying chromosomal disruptions of the genes encoding the mitochondrial C(1)-tetrahydrofolate (C(1)-THF) synthase (MIS1), necessary for synthesis of 10-formyl-THF, and the methionyl-tRNA formyltransferase (open reading frame YBL013W; designated FMT1). A direct analysis of mitochondrial tRNAs using gel electrophoresis systems that can separate fMet-tRNA(fMet), Met-tRNA(fMet), and tRNA(fMet) shows that there is no formylation in vivo of the mitochondrial initiator Met-tRNA in these strains. In contrast, the initiator Met-tRNA is formylated in the respective "wild-type" parental strains. In spite of the absence of fMet-tRNA(fMet), the mutant strains exhibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal frequencies of generation of petite colonies. The only growth phenotype observed was a longer lag time during growth on nonfermentable carbon sources in minimal media for the mis1 deletion strain but not for the fmt1 deletion strain.     

Anticodon sequence mutants of Escherichia coli initiator tRNA: effects of overproduction of aminoacyl-tRNA synthetases,methionyl-tRNA formyltransferase,and initiation factor 2 on activity in initiation

Anticodon sequence mutants of Escherichia coli initiator tRNA initiate protein synthesis with codons other than AUG and amino acids other than methionine. Because the anticodon sequence is, in many cases, important for recognition of tRNAs by aminoacyl-tRNA synthetases, the mutant tRNAs are aminoacylated in vivo with different amino acids. The activity of a mutant tRNA in initiation in vivo depends on (i) the level of expression of the tRNA, (ii) the extent of aminoacylation of the tRNA, (iii) the extent of formylation of the aminoacyl-tRNA to formylaminoacyl-tRNA (fAA-tRNA), and (iv) the affinity of the fAA-tRNA for the initiation factor IF2 and the ribosome. Previously, using E. coli overproducing aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, or IF2, we identified the steps limiting the activity in initiation of mutant tRNAs aminoacylated with glutamine and valine. Here, we have identified the steps limiting the activity of mutant tRNAs aminoacylated with isoleucine and phenylalanine. The combined results of experiments involving a variety of initiation codons (AUG, UAG, CAG, GUC, AUC, and UUC) provide support to the hypothesis that the ribosome.fAA-tRNA complex can act as an intermediate in initiation of protein synthesis. Comparison of binding affinities of various fAA-tRNAs (fMet-, fGln-, fVal-, fIle-, and fPhe-tRNAs) to IF2 using surface plasmon resonance supports the idea that IF2 can act as a carrier of fAA-tRNA to the ribosome. Other results suggest that the C1xA72 base pair mismatch, unique to eubacterial and organellar initiator tRNAs, may also be important for the binding of fAA-tRNA to IF2.     

Importance of the anticodon sequence in the aminoacylation of tRNAs by methionyl-tRNA synthetase and by valyl-tRNA synthetase in an Archaebacterium

The mode of recognition of tRNAs by aminoacyl-tRNA synthetases and translation factors is largely unknown in archaebacteria. To study this process, we have cloned the wild type initiator tRNA gene from the moderate halophilic archaebacterium Haloferax volcanii and mutants derived from it into a plasmid capable of expressing the tRNA in these cells. Analysis of tRNAs in vivo show that the initiator tRNA is aminoacylated but is not formylated in H. volcanii. This result provides direct support for the notion that protein synthesis in archaebacteria is initiated with methionine and not with formylmethionine. We have analyzed the effect of two different mutations (CAU-->CUA and CAU-->GAC) in the anticodon sequence of the initiator tRNA on its recognition by the aminoacyl-tRNA synthetases in vivo. The CAU-->CUA mutant was not aminoacylated to any significant extent in vivo, suggesting the importance of the anticodon in aminoacylation of tRNA by methionyl-tRNA synthetase. This mutant initiator tRNA can, however, be aminoacylated in vitro by the Escherichia coli glutaminyl-tRNA synthetase, suggesting that the lack of aminoacylation is due to the absence in H. volcanii of a synthetase, which recognizes the mutant tRNA. Archaebacteria lack glutaminyl-tRNA synthetase and utilize a two-step pathway involving glutamyl-tRNA synthetase and glutamine amidotransferase to generate glutaminyl-tRNA. The lack of aminoacylation of the mutant tRNA indicates that this mutant tRNA is not a substrate for the H. volcanii glutamyl-tRNA synthetase. The CAU-->GAC anticodon mutant is most likely aminoacylated with valine in vivo. Thus, the anticodon plays an important role in the recognition of tRNA by at least two of the halobacterial aminoacyl-tRNA synthetases.     

Utilization of an Escherichia coli mutant for carbon-13 enrichment of tRNA for NMR studies.

The enrichment of tRNA at specific sites with carbon-13 has been accomplished in vivo using a mutant of Escherichia coli. A relaxed strain of E. coli auxotrophic for methionine was grown in a specifically defined medium supplemented with either [14C] or [13C]-methyl labeled methionine. Cells were collected at the end of the log-phase of growth and tRNA was extracted. Analysis of the radioactivity of the [14C]-labeled tRNA established an incorporation ratio of three labeled carbons per tRNA molecule. Incorporation of the [14C]-label in vivo was confined to the methylation of nucleotides as determined by thin layer chromatography of nucleotides resulting from a ribonuclease digestion of [14C]-labeled tRNA. The carbon-13 NMR spectrum of [13C]-enriched tRNA indicated a similar degree of incorporation into the methylated nucleotides by the substantial enhancement of [13C]-methyl NMR signals only. Assignment of signals has been made for the methyl groups of ribothymidine and N7-methylguanosine in E. coli tRNA.     

Identification of bacteriophage T4-specific precursor tRNA by using a host mutant defective in the methylation of tRNA.

A mutant of Escherichia coli K-12 that is defective in the synthesis of 5-methyluridine (ribothymidine) in tRNA was used to identify precursors to phage T4-specific tRNA. The precursor molecules, isolated by gel filtration, were more than twice the size of tRNA. This method is suitable for isolation of rather large amounts of such precursor molecules.     

Formylatable methionine transfer RNA from Mycoplasma: purification and comparison of partial nucleotide sequences with those of other prokaryotic initiator tRNAs.

The major species of the formylatable methionine tRNA from Mycoplasma mycoides var capri has been purified. The 5'- and 3'-terminal sequences of the purified tRNA are pC-G- and C-A-A-C-C-AOH, respectively. Thus, this tRNA also contains the unique structural feature found in two other prokaryotic initiator tRNAs in that the first nucleotide at the 5'-end cannot form a Watson-Crick type of base-pair to the fifth nucleotide from the 3'-end. The Mycoplasma tRNA does not contain ribothymidine; however, a specific uridine residue in the sequence G-U-psi-C-G- can be enzymatically methylated by E. coli extracts to yield G-T-psi-C-G. Since ribothymidine is absent in crude tRNA from this strain of Mycoplasma, the absence of T is probably due to the lack of a U yields T modifying enzyme.     

Wheat germ tRNAs containing uridine in place of ribothymidine: a characterization of an unusual class of eukaryotic tRNAs.

An unusual class of wheat germ tRNAs has been isolated which completely lacks ribothymidine (rT) and contains an unmodified uridine in its place. We discuss here the isolation, identification and properties of these tRNAs. The rT-lacking tRNAs of wheat germ are essentially limited to the glycine isoacceptors (a minimum of five identifiable species), three threonine and at least, one tyrosine tRNA. All tRNAs were obtained 70-100% pure by chromatographic methods, and were detected by their ability to be methylated by E. coli rT-forming uracil methyltransferase with methyl-labeled S-adenosyl-L-methionine (SAM) as the methyl donor. In vitro methylation of each of the tRNAs resulted in the formation of 1 mole of rT per mole of tRNA. In the one case analyzed in detail (tRNA1Gly), all of the rT was found to be located at the 23rd position from the 3' end of the tRNA molecule. Following complete digestion of four highly purified glycine isoacceptors (tRNAGly1,4,5,6) to nucleosides and subsequent periodate oxidation and 3H potassium borohydride reduction, all were found to contain an unusually high level of 5-methylcytidine (m5C) (3-4 residues per molecule), and all contained no rT. The possible correlation between the presence of m5C and the absence of rT is discussed. All of the chromatographically purified glycine tRNAs function in a wheat germ cell-free protein synthesizing system and polymerize glycine in response to either poly G or poly (G, U).     

Initiation of Protein Synthesis in Mammalian Cells with Codons Other Than AUG and Amino Acids Other Than Methionine

Protein synthesis is initiated universally with the amino acid methionine. In Escherichia coli, studies with anticodon sequence mutants of the initiator methionine tRNA have shown that protein synthesis can be initiated with several other amino acids. In eukaryotic systems, however, a yeast initiator tRNA aminoacylated with isoleucine was found to be inactive in initiation in mammalian cell extracts. This finding raised the question of whether methionine is the only amino acid capable of initiation of protein synthesis in eukaryotes. In this work, we studied the activities, in initiation, of four different anticodon sequence mutants of human initiator tRNA in mammalian COS1 cells, using reporter genes carrying mutations in the initiation codon that are complementary to the tRNA anticodons. The mutant tRNAs used are aminoacylated with glutamine, methionine, and valine. Our results show that in the presence of the corresponding mutant initiator tRNAs, AGG and GUC can initiate protein synthesis in COS1 cells with methionine and valine, respectively. CAG initiates protein synthesis with glutamine but extremely poorly, whereas UAG could not be used to initiate protein synthesis with glutamine. We discuss the potential applications of the mutant initiator tRNA-dependent initiation of protein synthesis with codons other than AUG for studying the many interesting aspects of protein synthesis initiation in mammalian cells.     

Formylation is not essential for initiation of protein synthesis in all eubacteria.

Formylation of the initiator methionyl-tRNA, catalyzed by methionyl-tRNA formyltransferase, has long been regarded as essential for initiation of protein synthesis in eubacteria. Here, we show that this process is, in fact, dispensable in Pseudomonas aeruginosa. Disruption of the chromosomal methionyl-tRNA formyltransferase gene in P. aeruginosa resulted only in a moderate decrease in the rate of cell growth, whereas in Escherichia coli cell growth was severely impaired. The ability of the P. aeruginosa mutant strain to grow was not due to an additional copy of the methionyl-tRNA formyltransferase gene or to N-acylation of the methionyl moiety by a group other than formyl. These results indicate that P. aeruginosa can carry out formylation-independent initiation of protein synthesis, using the nonformylated methionyl-tRNA. Therefore, the dogma that eubacteria require formylation of the initiator methionyl-tRNA for initiation of protein synthesis may have been an invalid generalization of results obtained with E. coli.     

Requirement for IscS in biosynthesis of all thionucleosides in Escherichia coli

Escherichia coli tRNA contains four naturally occurring nucleosides modified with sulfur. Cysteine is the intracellular sulfur source for each of these modified bases. We previously found that the iscS gene, a member of the nifS cysteine desulfurase gene family, is required for 4-thiouridine biosynthesis in E. coli. Since IscS does not bind tRNA, its role is the mobilization and distribution of sulfur to enzymes that catalyze the sulfur insertion steps. In addition to iscS, E. coli contains two other nifS homologs, csdA and csdB, each of which has cysteine desulfurase activity and could potentially donate sulfur for thionucleoside biosynthesis. Double csdA csdB and iscS csdA mutants were prepared or obtained, and all mutants were analyzed for thionucleoside content. It was found that unfractionated tRNA isolated from the iscS mutant strain contained <5% of the level of sulfur found in the parent strain. High-pressure liquid chromatography analysis of tRNA nuclease digests from the mutant strain grown in the presence of [(35)S]cysteine showed that only a small fraction of 2-thiocytidine was present, while the other thionucleosides were absent when cells were isolated during log phase. As expected, digests from the iscS mutant strain contained 6-N-dimethylallyl adenosine (i(6)A) in place of 6-N-dimethylallyl-2-methylthioadenosine and 5-methylaminomethyl uridine (mnm(5)U) instead of 5-methylaminomethyl-2-thiouridine. Prolonged growth of the iscS and iscS csdA mutant strains revealed a gradual increase in levels of 2-thiocytidine and 6-N-dimethylallyl-2-methylthioadenosine with extended incubation (>24 h), while the thiouridines remained absent. This may be due to a residual level of Fe-S cluster biosynthesis in iscS deletion strains. An overall scheme for thionucleoside biosynthesis in E. coli is discussed.     

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.     

Nucleotides of tRNA governing the specificity of Escherichia coli methionyl-tRNA(fMet) formyltransferase.

In Escherichia coli, the free amino group of the aminoacyl moiety of methionyl-tRNA(fMet) is specifically modified by a transformylation reaction. To identify the nucleotides governing the recognition of the tRNA substrate by the formylase, initiator tRNA(fMet) was changed into an elongator tRNA with the help of an in vivo selection method. All the mutations isolated were in the tRNA acceptor arm, at positions 72 and 73. The major role of the acceptor arm was further established by the demonstration of the full formylability of a chimaeric tRNA(Met) containing the acceptor stem of tRNA(fMet) and the remaining of the structure of tRNA(mMet). In addition, more than 30 variants of the genes encoding tRNA(mMet) or tRNA(fMet) have been constructed, the corresponding mutant tRNA products purified and the parameters of the formylation reaction measured. tRNA(mMet) became formylatable by the only change of the G1.C72 base-pair into C1-A72. It was possible to render tRNA(mMet) as good a substrate as tRNA(fMet) for the formylase by the introduction of a limited number of additional changes in the acceptor stem. In conclusion, A73, G2.C71, C3.G70 and G4.C69 are positive determinants for the specific processing of methionyl-tRNA(fMet) by the formylase while the occurrence of a G.C or C.G base-pair between positions 1 and 72 acts as a major negative determinant. This pattern appears to account fully for the specificity of the formylase and the lack of formylation of any aminoacylated tRNA, excepting the methionyl-tRNA(fMet).     

Tetrahydrofolate-dependent biosynthesis of ribothymidine in transfer ribonucleic acids of Gram-positive bacteria.

Trimethoprim, an inhibitor that prevents tetrahydrofolate-dependent transmethylation reactions inbacteria, was used in a comparative study to discriminate between two possible biosynthetic pathways, either the S-adenosylmethionine or the tetrahydrofolate-dependent formation of ribothymidine (rT) in transfer ribonucleic acids (tRNA's) of several strains of gram-positive and gram-negative microorganisms. rT-deficient tRNA's accumulate in trimethoprim-treated gram-positive Streptococcus faecium, Staphylococcus aureus, Corynebacterium bovis, Arthrobacter albidus, and all examined Bacillaceae, except Bacillus stearothermophilus. The rT-deficient rT-deficient tRNA's accept the methyl moiety from S-adenosylmethionine in vitro, with extracts from Escherichia coli (wild type) as a source of methylating enzymes; 90% of the incorporated methyl groups are present in rT. Trimethoprim does not inhibit the biosynthesis of rT in tRNA of gram-negative Enterobacteriaceae, Rhizobium lupini, and Pseudomonadaceae, suggesting that the rT-specific tRNA methyltransferases of these gram-negative strains use S-adenosylmethionine as coenzyme.     

Initiation of in vivo protein synthesis with non-methionine amino acids

Methionine is the universal amino acid for initiation of protein synthesis in all known organisms. The amino acid is coupled to a specific initiator methionine tRNA by methionyl-tRNA synthetase. In Escherichia coli, attachment of methionine to the initiator tRNA (tRNA(fMet)) has been shown to be dependent on synthetase recognition of the methionine anticodon CAU (complementary to the initiation codon AUG), [Schulman, L. H., & Pelka, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6755-6759]. We show here that alteration of the anticodon of tRNA(fMet) to GAC or GAA leads to aminoacylation of the initiator tRNA with valine or phenylalanine. In addition, tRNA(fMet) carrying these amino acids initiates in vivo protein synthesis when provided with initiation codons complementary to the modified anticodons. These results indicate that the sequence of the anticodon of tRNA(fMet) dictates the identity of the amino acid attached to the initiator tRNA in vivo and that there are no subsequent steps which prevent initiation of E. coli protein synthesis by valine and phenylalanine. The methods described here also provide a convenient in vivo assay for further examination of the role of the anticodon in tRNA amino acid acceptor identity.     

Function of Modified Nucleosides 7-Methylguanosine, Ribothymidine, and 2-Thiomethyl-N6-(Isopentenyl)adenosine in Procaryotic Transfer Ribonucleic Acid

To elucidate subtle functions of transfer ribonucleic acid (tRNA) modifications in protein synthesis, pairs of tRNA's that differ in modifications at specific positions were prepared from Bacillus subtilis. The tRNA's differ in modifications in the anticodon loop, the extra arm, and the TUC loop. The functional properties of these species were compared in aminoacylation, as well as in initiation and peptide bond formation, at programmed ribosomes. These experiments demonstrated the following. (i) In tRNA(f) (Met) the methylation of guanosine 46 in the extra arm to 7-methylguanosine by the 7-methylguanosine-forming enzyme from Escherichia coli changes the aminoacylation kinetics for the B. subtilis methionyl-tRNA synthetase. In repeated experiments the V(max) value is decreased by one-half. (ii) tRNA(f) (Met) species with ribothymidine at position 54 (rT54) or uridine at position 54 (U54) were obtained from untreated or trimethoprim-treated B. subtilis. The formylated fMet-tRNA(f) (Met) species with U54 and rT54, respectively, function equally well in an in vitro initiation system containing AUG, initiation factors, and 70s ribosomes. The unformylated Met-tRNA(t) (Met) species, however, differ from each other: "Met-tRNA(f) (Met) rT" is inactive, whereas the U54 counter-upart effectively forms the initiation complex. (iii) Two isoacceptors, tRNA(1) (Phe) and tRNA(2) (Phe), were obtained from B. subtilis. tRNA(1) (Phe) accumulates only under special growth conditions and is an incompletely modified precursor oftRNA(2) (Phe): in the first position of the anticodon, guanosine replaces Gm, and next to the 3' end of the anticodon (isopentenyl)adenosine replaces 2-thiomethyl-N(6)-(isopentenyl)adenosine. Both tRNA's behave identically in aminoacylation kinetics. In the factor-dependent AUGU(3)-directed formation of fMet-Phe, the undermodified tRNA(1) (Phe) is always less efficient at Mg(2+) concentrations between 5 and 15 mM than its mature counterpart.