Initiation with Elongator tRNAs

In all domains of life, initiator tRNA functions exclusively at the first step of protein synthesis while elongator tRNAs extend the polypeptide chain. Unique features of initiator tRNA enable it to preferentially bind the ribosomal P site and initiate translation. Recently, we showed that the abundance of initiator tRNA also contributes to its specialized role. This motivates the question, can a cell also use elongator tRNA to initiate translation under certain conditions? To address this, we introduced non-AUG initiation codons CCC (Pro), GAG (Glu), GGU (Gly), UCU (Ser), UGU (Cys), ACG (Thr), AAU (Asn), and AGA (Arg) into the uracil DNA glycosylase gene (ung) used as a reporter gene. Enzyme assays from log-phase cells revealed initiation from non-AUG codons when intracellular initiator tRNA levels were reduced. The activity increased significantly in stationary phase. Further increases in initiation from non-AUG codons occurred in both growth phases upon introduction of plasmid-borne genes of cognate elongator tRNAs. Since purine-rich Shine-Dalgarno sequences occur frequently on mRNAs (in places other than the canonical AUG codon initiation contexts), initiation with elongator tRNAs from the alternate contexts may generate proteome diversity under stress without compromising genomic integrity. Thus, by changing the relative amounts of initiator and elongator tRNAs within the cell, we have blurred the distinction between the two classes of tRNAs thought to be frozen through years of evolution.     

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).     

Analysis of magnesium, europium and lead binding sites in methionine initiator and elongator tRNAs by specific metal-ion-induced cleavages

The specificity of cleavages in yeast and lupin initiator and elongator methionine tRNAs induced by magnesium, europium and lead has been analysed and compared with known patterns of yeast tRNA(Phe) hydrolysis. The strong D-loop cleavages occur in methionine elongator tRNAs at similar positions and with comparable efficiency to those found in tRNA(Phe), while the sites of weak anticodon loop cuts, identical in methionine elongator tRNAs, differ from those found in tRNA(Phe). Methionine initiator tRNAs differ from their elongator counterparts: (a) they are cleaved in the D-loop with much lower efficiency; (b) they are cleaved in the variable loop which is completely resistant to hydrolysis in elongator tRNAs; (c) cleavages in the anticodon loop are stronger in initiator tRNAs and they are located mostly at the 5' side of the loop whereas in elongator tRNAs they occur mostly at the opposite, 3' side of the loop. The distinct pattern of the anticodon loop cleavages is considered to be related to different conformations of the anticodon loop in the two types of methionine tRNAs.     

Sequence and structure of a methionine transfer RNA from mosquito mitochondria.

We have sequenced a methionine tRNA from mosquito mitochondria, and examined its structure using nucleases S1 and T1 under non-denaturing conditions. The sequence is highly homologous to a putative initiator methionine tRNA gene from Drosophila mitochondria. Its anticodon stem contains a run of three G-C base pairs that is characteristic of conventional initiator tRNAs; however, nuclease S1 analysis suggested an anticodon loop configuration characteristic of conventional elongator tRNAs. We propose that this tRNA can assume both initiator and elongator roles.     

Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis

Initiator tRNAs are used exclusively for initiation of protein synthesis and not for the elongation step. We show, in vivo and in vitro, that the primary sequence feature that prevents the human initiator tRNA from acting in the elongation step is the nature of base pairs 50:64 and 51:63 in the TΨC stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the TΨC stem of initiator tRNA, which most likely blocks binding of the elongation factor to the tRNA. Because the sequences of all vertebrate initiator tRNAs are identical, our findings with the human initiator tRNA are likely to be valid for all vertebrate systems. We have developed reporter systems that can be used to monitor, in mammalian cells, the activity in elongation of mutant human initiator tRNAs carrying anticodon sequence mutations from CAU to CCU (the C35 mutant) or to CUA (the U35A36 mutant). Combination of the anticodon sequence mutation with mutations in base pairs 50:64 and 51:63 yielded tRNAs that act as elongators in mammalian cells. Further mutation of the A1:U72 base pair, which is conserved in virtually all eukaryotic initiator tRNAs, to G1:C72 in the C35 mutant background yielded tRNAs that were even more active in elongation. In addition, in a rabbit reticulocyte in vitro protein-synthesizing system, a tRNA carrying the TΨC stem and the A1:U72-to-G1:C72 mutations was almost as active in elongation as the elongator methionine tRNA. The combination of mutant initiator tRNA with the CCU anticodon and the reporter system developed here provides the first example of missense suppression in mammalian cells.     

Structural features that underlie the use of bacterial Met-tRNAfMet primarily as an elongator in eukaryotic protein synthesis

Met-tRNAfMet from Escherichia coli is utilized efficiently as an elongator tRNA during protein synthesis in the rabbit reticulocyte lysate since it rapidly incorporates its methionyl residue into the same tryptic peptides of rabbit globin as the endogenous Met-tRNAmMet. Therefore, it must lack the structural characteristics that prevent the eukaryotic initiator tRNA from entering elongation. In contrast, E. coli Met-tRNAfMet appears to initiate very poorly since, unlike reticulocyte Met-tRNAiMet, it forms no detectable 43 S preinitiation complexes, and only a very small fraction of the methionine it contributes to polyribosomal peptidyl-tRNA is found at the N terminus. The bacterial fMet-tRNAfMet, which cannot elongate, is utilized for polypeptide chain initiation at a much lower level than the formylated Met-tRNAiMet from eukaryotes. The ability of E. coli Met-tRNAfMet to be used as an elongator and fMet-tRNAfMet as an initiator in the reticulocyte lysate may be considerably underestimated because of the rapid enzymatic hydrolysis of these initiator tRNAs in the lysate. The enzyme hydrolyzes fMet-tRNAfMet and Met-tRNAfMet from E. coli in a strictly Mg2+-dependent manner but not the corresponding species from yeast or rabbit reticulocytes. It also hydrolyzes yeast N-acetyl-Phe-tRNAPhe and reticulocyte peptidyl-tRNA, showing that this enzyme--like the eukaryotic protein synthetic machinery--does not readily distinguish the bacterial tRNAfMet from eukaryotic elongator tRNA.     

A unique conformation of the anticodon stem-loop is associated with the capacity of tRNAfMet to initiate protein synthesis

In all organisms, translational initiation takes place on the small ribosomal subunit and two classes of methionine tRNA are present. The initiator is used exclusively for initiation of protein synthesis while the elongator is used for inserting methionine internally in the nascent polypeptide chain. The crystal structure of Escherichia coli initiator tRNA(f)(Met) has been solved at 3.1 A resolution. The anticodon region is well-defined and reveals a unique structure, which has not been described in any other tRNA. It encompasses a Cm32*A38 base pair with a peculiar geometry extending the anticodon helix, a base triple between A37 and the G29-C41 pair in the major groove of the anticodon stem and a modified stacking organization of the anticodon loop. This conformation is associated with the three GC basepairs in the anticodon stem, characteristic of initiator tRNAs and suggests a mechanism by which the translation initiation machinery could discriminate the initiator tRNA from all other tRNAs.     

Electrical potential of transfer RNAs: codon-anticodon recognition

Calculations of the electrostatic potentials were made around yeast elongator phenylalanine, aspartate tRNAs, and yeast initiator methionine tRNA in aqueous solution at physiological ionic strength. The calculations were carried out with a finite difference algorithm for solving the nonlinear Poisson-Boltzmann equation that incorporates the screening effects of the electrolyte, the exclusion of ions by the molecule, the molecular shape, and the different polarizabilities of the solvent and the tRNA. The initiator tRNA is surrounded by uniformly spaced contours of negative potential. The elongator tRNAs are also surrounded by a similar contour pattern except in the anticodon region where there is a pronounced "hole" in the potential surface. This hole is caused by an invagination of the potential contours, which also results in an increase in the local field strength. The effect of this hole is that the anticodon region in the elongator tRNAs is the least negative, or conversely the most positive, region of the molecule. This hole, which is not found when simple Coulombic potentials are used, is due both to the structure of the elongator tRNA anticodon loops and to the different polarizabilities of the solvent and tRNA. The existence of the potential hole in elongator tRNAs may account in part for their ability to associate with other negatively charged macromolecules, in particular mRNA. Moreover, it suggests that the anticodon loop of elongator tRNAs is the energetically most favorable point of approach of mRNA to tRNA.     

The T-stem determines the cytosolic or mitochondrial localization of trypanosomal tRNAsMet

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Organellar translation therefore depends on import of cytosolic, nucleus-encoded tRNAs. Except for the cytosol-specific initiator tRNA(Met), all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The initiator tRNA(Met) is closely related to the imported elongator tRNA(Met). Thus, the distinct localization of the two tRNAs(Met) must be specified by the 26 nucleotides, which differ between the two molecules. Using transgenic T. brucei cell lines and subsequent cell fractionation, we show that the T-stem is both required and sufficient to specify the localization of the tRNAs(Met). Furthermore, it was shown that the tRNA(Met) T-stem localization determinants are also functional in the context of two other tRNAs. In vivo analysis of the modified nucleotides found in the initiator tRNA(Met) indicates that the T-stem localization determinants do not require modified nucleotides. In contrast, import of native tRNAs(Met) into isolated mitochondria suggests that nucleotide modifications might be involved in regulating the extent of import of elongator tRNA(Met).     

Interaction between initiator Met-tRNAfMet and elongation factor EF-Tu from E. coli

It has recently been shown that the non-formylated initiator Met-tRNAfMet from E. coli can form a stable ternary complex with the elongation factor EF-Tu and GTP. Using the protection of EF-Tu:GTP against spontaneous hydrolysis of the aminoacylester bond of Met-tRNAfMet, we confirm these results, and show that the protection is specific for the non-formylated form of the initiator tRNA. The ternary complex Met-tRNAfMet:EF-Tu:GTP can be isolated by column chromatography in a way similar to that demonstrated previously with EF-Tu complexed to the elongator Met-tRNAmMet. 32P-labeled Met-tRNAfMet within the ternary complex was analyzed by the footprinting technique. The pattern of initiator tRNA protection by EF-Tu against ribonuclease digestion is not significantly different from the one found previously for elongator tRNAs. These results lead us to suggest that the initiator tRNAfMet, under growth conditions which do not permit formylation, may to some extent function as an elongator tRNA.     

Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.

The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.     

28 The study of tRNA modifications by molecular dynamics

Modified nucleosides are prevalent in tRNA. Experimental studies reveal that modifications play an important role in tuning tRNA activity. In this study, molecular dynamics (MD) simulations were used to investigate how modifications alter tRNA structure and dynamics. The X-ray crystal structures of tRNA-Asp, tRNA-Phe, and tRNA-iMet, both with and without modifications, were used as initial structures for 333-ns time-scale MD trajectories with AMBER. For each tRNA molecule, three independent trajectory calculations were performed. Force field parameters were built using the RESP procedure of Cieplak et al. for 17 nonstandard tRNA residues. The global root-mean-square deviations (RMSDs) of atomic positions show that modifications only introduce significant rigidity to tRNA-Phe’s global structure. Interestingly, regional RMSDs of anticodon stem-loop suggest that modified tRNA has more rigid structure compared to the unmodified tRNA in this domain. The anticodon RMSDs of the modified tRNAs, however, are higher than those of corresponding unmodified tRNAs. These findings suggest that rigidity of the anticodon arm is essential for tRNA translocation in the ribosome complex, and, on the other hand, flexibility of anticodon might be critical for anticodon–codon recognition. We also measure the angle between the 3D L-shaped arms of tRNA; backbone atoms of acceptor stem and TψC stem loop are selected to indicate one vector, and backbone atoms of anticodon stem and D stem loop are selected to indicate the other vector. By measuring the angle between two vectors, we find that the initiator tRNA has a narrower range of hinge motion compared to tRNA-Asp and tRNA-Phe, which are elongator tRNA. This suggests that elongator tRNAs, which might require significant flexibility in this hinge to transition from the A–to-P site in the ribosome, have evolved to specifically accommodate this need.     

Suppression of amber codons in vivo as evidence that mutants derived from Escherichia coli initiator tRNA can act at the step of elongation in protein synthesis

The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs. We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo. We generated a mutant of E. coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant). This mutant tRNA has the potential to read the amber termination codon UAG. We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant). Transformation of E. coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not. Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro. Measurement of kinetic parameters for aminoacylation by E. coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics. We discuss the implications of this result on recognition of tRNAs by E. coli glutaminyl-tRNA synthetase.     

Nucleotide sequence of cytoplasmic initiator tRNA from Tetrahymena thermophila.

The total primary structure of cytoplasmic initiator tRNA from Tetrahymena thermophila mating type IV, was determined by post labeling techniques. The sequence is pa-G-C-A-G-G-G-U-m1G-G-C-G-A-A-A-D-Gm-G-A-A-U-C-G-C-G-U-Psi-G-G-G-C-U-C-A-U-t6A -A-C-Psi-C-A-A-A-A-m7G-U-m5C-A-G-A-G-G-A-Psi-C-G-m1A-A-A-C-C-U-C-U-C-U-C-U-G-C- U-A-C-C-AOH. The nucleotide residue in the position next to the 5'-end of the anticodon of this tRNA (residue No. 33) is uridine instead of cytidine, which has been found in cytoplasmic initiator tRNAs from multicellular eukaryotic organisms. The sequence of three consecutive G-C base pairs in the anticodon stem common to all other cytoplasmic initiator tRNAs is disrupted in this tRNA; namely, the cytidine at residue 40 in this region is replaced by pseudouridine in Tetrahymena initiator tRNA.     

The nucleotide sequence of formylmethionine tRNA from Mycoplasma mycoides sp. capri.

The nucleotide sequence of Mycoplasma mycoides sp. capri PG3 formylmethionine tRNA has been determined, using in vitro labeling techniques, to be pC-G-C-G-G-G-G-s4U-A-G-A-G-C-A-G-U-D (U)-G-G-D-A-G-C-U-C-G-C-C-G-G-G-C-U-C-A-U-A-A-C-C-C-G-G-A-G-G-C-C-G-C-A-G-G-U-psi- C-G-A-G-U-C-C-U-G-C-C-C-C-C-G-C-A-A-C-C-AOH. This tRNA contains only three modified nucleosides s4U, D and psi, all of which are derived from uridine. Both in the structural features which distinguish eukaryotic from prokaryotic initiator RNAs and in the overall sequence, this tRNA resembles a typical prokaryotic initiator tRNA. A comparison of the sequence of this tRNA with those of other prokaryotic initiator tRNAs suggests that taxonomically the Mycoplasma may be less related to the Cyanophyta (Anacystis nidulans) than to the bacteria and less related to the Enterobacteriaceae (Escherichia coli) than to the Bacillaceae (Bacillus subtilis).     

A synthetic alanyl-initiator tRNA with initiator tRNA properties as determined by fluorescence measurements: comparison to a synthetic alanyl-elongator tRNA.

Two synthetic tRNAs have been generated that can be enzymatically aminoacylated with alanine and have AAA anticodons to recognize a poly(U) template. One of the tRNAs (tRNA(eAla/AAA)) is nearly identical to Escherichia coli elongator tRNA(Ala). The other has a sequence similar to Escherichia coli initiator tRNA(Met) (tRNA(iAla/AAA)). Although both tRNAs can be used in poly(U)-directed nonenzymatic initiation at 15 mM Mg2+, only the elongator tRNA can serve for peptide elongation and polyalanine synthesis. Only the initiator tRNA can be bound to 30S ribosomal subunits or 70S ribosomes in the presence of initiation factor 2 (IF-2) and low Mg2+ suggesting that it can function in enzymatic peptide initiation. A derivative of coumarin was covalently attached to the alpha amino group of alanine of these two Ala-tRNA species. The fluorescence spectra, quantum yield and anisotropy for the two Ala-tRNA derivatives are different when they are bound to 70S ribosomes (nonenzymatically in the presence of 15 mM Mg2+) indicating that the local environment of the probe is different. Also, the effect of erythromycin on their fluorescence is quite different, suggesting that the probes and presumably the alanine moiety to which they are covalently linked are in different positions on the ribosomes.     

Effects of magnesium and ionic strength on the diffusion and charge properties of several single tRNA species.

The technique of laser light scattering was used to evaluate the effects of Mg+2 and ionic strength on the solution structures of seven tRNA species. Information about ion effects on both conformation and electric charge were derived from measurements of the translational diffusion constants and diffusive virial coefficients. E. coli tRNAMetf and six elongator tRNAs from both Class I and II were studied. The diffusion measurements show that the responses of all but the initiator species are qualitatively similar to each other and to that of bulk tRNA, but that significant quantitative differences also obtain. All of the elongator species exhibited an anomolous increase in diffusivity reported earlier by us for bulk tRNA when placed in a low salt-low Mg+2 condition. The initiator tRNA did not undergo this transition and unlike the other tRNAs tested was apparently more compact in 1 mM Mg+2 than 10 mM Mg+2 at ionic strengths in excess of 0.1 M. At 0.1 M ionic strength, pH 7.2, the average net charge of the tRNAs ranged from 7-12 e- in 1 mM Mg+2 and 3-7 e- in 10 mM Mg+2, consistent with the binding of 1-2 additional Mg+2 ions in the higher Mg+2 condition.     

Eukaryotic initiator tRNA: Finely tuned and ready for action

The initiator tRNA must serve functions distinct from those of other tRNAs, evading binding to elongation factors and instead binding directly to the ribosomal P site with the aid of initiation factors. It plays a key role in decoding the start codon, setting the frame for translation of the mRNA. Sequence elements and modifications of the initiator tRNA distinguish it from the elongator methionyl tRNA and help it to perform its varied tasks. These identity elements appear to finely tune the structure of the initiator tRNA, and growing evidence suggests that the body of the tRNA is involved in transmitting the signal that the start codon has been found to the rest of the pre-initiation complex.     

Common location of determinants in initiator transfer RNAs for initiator-elongator discrimination in bacteria and in eukaryotes

Initiator tRNAs are used exclusively for initiation of protein synthesis and not for elongation. We show that both Escherichia coli and eukaryotic initiator tRNAs have negative determinants, at the same positions, that block their activity in elongation. The primary negative determinant in E. coli initiator tRNA is the C1xA72 mismatch at the end of the acceptor stem. The primary negative determinant in eukaryotic initiator tRNAs is located in the TPsiC stem, whereas a secondary negative determinant is the A1:U72 base pair at the end of the acceptor stem. Here we show that E. coli initiator tRNA also has a secondary negative determinant for elongation and that it is the U50.G64 wobble base pair, located at the same position in the TPsiC stem as the primary negative determinant in eukaryotic initiator tRNAs. Mutation of the U50.G64 wobble base pair to C50:G64 or U50:A64 base pairs increases the in vivo amber suppressor activity of initiator tRNA mutants that have changes in the acceptor stem and in the anticodon sequence necessary for amber suppressor activity. Binding assays of the mutant aminoacyl-tRNAs carrying the C50 and A64 changes to the elongation factor EF-Tu.GTP show marginally higher affinity of the C50 and A64 mutant tRNAs and increased stability of the EF-Tu.GTP. aminoacyl-tRNA ternary complexes. Other results show a large effect of the amino acid attached to a tRNA, glutamine versus methionine, on the binding affinity toward EF-Tu.GTP and on the stability of the EF-Tu.GTP.aminoacyl-tRNA ternary complex.     

The nucleotide sequence of a methionine tRNA which functions in protein elongation in mouse myeloma cells.

The major form of methionine tRNA operational in the elongation of protein synthesis in mouse myeloma cells was purufied from these cells after they had been cultured in the presence of [32P]-phosphate. This [32P]tRNA4-Met species was then digested with T1 RNase or pancreatic RNase so as to obtain both complete and partial RNase digestion products. The nucleotide sequences of these fragments were analysed to enable the derivation of the complete primary structure of this tRNA. tRNA4-Met of mouse myeloma cells is 76 nucleotides in length and contains 15 modified nucleotides. It is the only tRNA yet sequenced which has been found to possess the minor nucleoside 2-methylguanosine (m2G) within the amino acid (a) stem, and also to have an anticodon (c) stem of only 4 and not 5 base-pairs. The loop IV sequence of eukaryotic initiator methionine tRNA (tRNAf-Met) species, -A-U-C-G-m1A-A-A-, IS NOT FOUND IN TRNA4-Met and is therefore absent from at least one of the methionine tRNAs functioning in polypeptide elongation in mammalian cells. This is consistent with the suggested importance of this loop structure in the initiator function of tRNAf-Met in eukaryotic organisms. Three distinct regions of the tRNA cloverleaf, the (b) stem, the anticodon loop (loop II), and loop III, are substantially conserved in structure between tRNAf-Met and tRNA4-Met of mouse myeloma cells. These regions of the structures of mammalian methionine tRNAs probably do not determine whether a certain tRNA-Met will function in the initiation or elongation of protein synthesis, although they might be important in tRNA-Met recognition if the different cytoplasmic tRNA-Met species of mammalian cells are aminoacylated by a single activating enzyme.