Nucleotide sequence of wheat germ cytoplasmic initiator methionine transfer ribonucleic acid

The primary sequence of wheat germ initiator tRNA has been determined using in vitro labelling techniques. The sequence is: pAUCAGAGUm1Gm2GCGCAG CGGAAGCGUm2GG psi GGGCCCAUt6AACCCACAGm7GDm5Cm5CCAGGA psi CGm1AAACCUG*GCUCUGAUACCAOH. As in other eukaryotic initiator tRNAs, the sequence -T psi CG(A)- present in loop IV of virtually all tRNA active in protein synthesis is absent and is replaced by -A psi CG-. The base pair G2:C71 present in all other initiator tRNAs recognized by E. coli Met-tRNA transformylase is absent and is replaced by U2:A71. Since wheat germ initiator tRNA is not formylated by E. coli Met-tRNA transformylase this implies a possible role of the G2:C71 base pair present in other initiator tRNAs in formylation of initiator tRNA species.     

Nucleotide sequence of starfish initiator tRNA.

The nucleotide sequence of starfish ovary initiator tRNA was determined to be pA-G-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-U-G-G-A-A-G-C-G-U-G-C-U-G-G-G-C-C-C-A-U-t6A-A-C-C-C-A-G-A-G-m7G-D-m5C-C-G-A-G-G-A-psi-C-G-m1A-A-A-C-C-U-C-G-C-U-C-U-G-C-U-A-C-C-AOH. The sequence was determined by a combination of the two different post-labeling techniques. Two-dimensional cellulose thin-layer chromatography was adopted for analysis of 5'-terminal nucleotides of tRNA fragments produced by formamide treatment. The nucleotide sequence of starfish initiator tRNA is very similar to that of mammalian cytoplasmic initiator tRNAs, but has seven different nucleotide residues and two modifications: residue 55 is psi instead of U, and residue 26 is unmodified G instead of m2G.     

Assembly of the mitochondrial membrane system. Sequences of yeast mitochondrial tRNA genes

Two cytoplasmic "petite" (rho-) clones of Saccharomyces cerevisiae have been selected for the retention of the aspartic acid tRNA gene. The two clones, designated DS200/A102 and DS200/A5, have tandemly repeated segments of mitochondrial DNA (mtDNA) with unit lengths of 1,000 and 6,400 base pairs, respectively. The DS200/A102 genome has a single tRNA gene with a 3'-CUG-5' anticodon capable of recognizing the 5'-GAC-3' and 5'-GAU-3' codons for aspartic acid. The mtDNA segment of DS200/A102 has been determined to represent the wild type sequence from 5.3 to 6.8 map units. The genome of DS200/A5 is more complex encompassing the region of wild type mtDNA from 3.5 to 12.7 units. A continuous sequence has been obtained from 3.5 to 8.6 units. In addition to the aspartic acid tRNA, this region codes for the tRNAUGCAla,tRNAUCUArg, tRNAACGArg, tRNAGCUSer,tRNAUCCGly and tRNAUUULys. The DNA sequence of the DS200/A5 genome has allowed us to deduce the secondary structures of the seven tRNAs and to assign precise map positions for their genes. All the tRNAs except tRNA GUCAsp exhibit most of the invariant features of prokaryotic and eukaryotic tRNAs. The aspartic acid tRNA has unusual D and T psi C loops. The structure of this tRNA is similar to the mitochondrial initiator tRNA of Neurospora crassa (Heckman, J.E., Hecker, L.I., Shwartzbach, S.D., Barnett, W.E., Baumstark, B., and RajBhandary, U.L. Cell 13, 83-95).     

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.     

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.     

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).     

Structure and function of initiator methionine tRNA from the mitochondria of Neurospora crassa.

Initiator methionine tRNA from the mitochondria of Neurospora crassa has been purified and sequenced. This mitochondrial tRNA can be aminoacylated and formylated by E. coli enzymes, and is capable of initiating protein synthesis in E. coli extracts. The nucleotide composition of the mitochondrial initiator tRNA (the first mitochondrial tRNA subjected to sequence analysis) is very rich in A + U, like that reported for total mitochondrial tRNA. In two of the unique features which differentiate procaryotic from eucaryotic cytoplasmic initiator tRNAs, the mitochondrial tRNA appears to resemble the eucaryotic initiator tRNAs. Thus unlike procaryotic initiator tRNAs in which the 5′ terminal nucleotide cannot form a Watson-Crick base pair to the fifth nucleotide from the 3′ end, the mitochondrial tRNA can form such a base pair; and like the eucaryotic cytoplasmic initiator tRNAs, the mitochondrial initiator tRNA lacks the sequence -TΨCG(or A) in loop IV. The corresponding sequence in the mitochondrial tRNA, however, is -UGCA- and not -AU(or Ψ)CG-as found in all eucaryotic cytoplasmic initiator tRNAs. In spite of some similarity of the mitochondrial initiator tRNA to both eucaryotic and procaryotic initiator tRNAs, the mitochondrial initiator tRNA is basically different from both these tRNAs. Between these two classes of initiator tRNAs, however, it is more homologous in sequence to procaryotic (56–60%) than to eucaryotic cytoplasmic initiator tRNAs (45–51%).     

Rabbit liver tRNA1Val:II. unusual secondary structure of T psi C stem and loop due to a U54:A60 base pair.

In contrast to all other known tRNAs, mammalian tRNA1Val contains two adenosines A59 and A60, opposite to U54 and psi 55 in the U psi CG sequence of the T psi C loop, which could form unusual A:U (or A: psi pairs in addition to the five "normal" G:C pairs. In order to measure the number of G:C and A:U (A: psi) pairs in the T psi C stem, we prepared the 30 nucleotide long 3'-terminal fragment of this tRNA by "m7G-cleavage". From differentiated melting curves and temperature jump experiments it was concluded that the T psi C stem in this fragment is in fact extended by an additional A60:U54 pair. A dimer of this fragment with 14 base pairs was characterized by gel electrophoresis and by the same physical methods. An additional A:U pair in the tRNA1Val fragment does not necessarily mean that this is also true for intact tRNA. However, we showed that U54 is far less available for enzymatic methylation in mammalian tRNA1Val compared to tRNA from T-E. coli. This clear difference in U54 reactivity, together with the identification of an extra A60:U54 pair in the U psi CG containing fragment suggests the presence of a 6 base pair T psi C stem and a 5 nucleotide T psi C loop in this tRNA.     

The A1 x U72 base pair conserved in eukaryotic initiator tRNAs is important specifically for binding to the eukaryotic translation initiation factor eIF2.

The formation of a specific ternary complex between eukaryotic initiation factor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation initiation in the cytoplasmic protein-synthesizing system of eukaryotes. We show that the A1 x U72 base pair conserved at the end of the acceptor stem in eukaryotic and archaebacterial initiator methionine tRNAs plays an important role in this interaction. We changed the A1 x U72 base pair of the human initiator tRNA to G1 x C72 and expressed the wild-type and mutant tRNA genes in the yeast Saccharomyces cerevisiae by using constructs previously developed in our laboratory for expression of the human initiator tRNA gene in yeasts. We show that both the wild-type and mutant human initiator tRNAs are aminoacylated well in vivo. We have isolated the wild-type and mutant human initiator tRNAs in substantially pure form, free of the yeast initiator tRNA, and have analyzed their properties in vitro. The G1 x C72 mutation affects specifically the binding affinity of eIF2 for the initiator tRNA. It has no effect on the subsequent formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the puromycin reactivity of the Met-tRNA in the 80S initiation complex.     

Yeast mitochondrial methionine initiator tRNA: characterization and nucleotide sequence.

Two methionine tRNAs from yeast mitochondria have been purified. The mitochondrial initiator tRNA has been identified by formylation using a mitochondrial enzyme extract. E. coli transformylase however, does not formylate the yeast mitochondrial initiator tRNA. The sequence was determined using both 32P-in vivo labeled and 32P-end labeled mt tRNAf(Met). This tRNA, unlike N. crassa mitochondrial tRNAf(Met), has two structural features typical of procaryotic initiator tRNAs: (i) it lacks a Watson-Crick base-pair at the end of the acceptor stem and (ii) has a T-psi-C-A sequence in loop IV. However, both yeast and N. crassa mitochondrial initiator tRNAs have a U11:A24 base-pair in the D-stem unlike procaryotic initiator tRNAs which have A11:U24. Interestingly, both mitochondrial initiator tRNAs, as well as bean chloroplast tRNAf(Met), have only two G:C pairs next to the anticodon loop, unlike any other initiator tRNA whatever its origin. In terms of overall sequence homology, yeast mitochondrial tRNA(Met)f differs from both procaryotic or eucaryotic initiator tRNAs, showing the highest homology with N. crassa mitochondrial initiator tRNA.     

500-MHz proton homonuclear Overhauser evidence for additional base pair in the common arm of eukaryotic ribosomal 5S RNA: wheat germ

A "common-arm" fragment from wheat germ (Triticum aestivum) 5S RNA has been produced by enzymatic cleavage with RNase T1 and sequenced via autoradiography of electrophoresis gels for the end-labeled fragments obtained by further RNase T1 partial digestion. The existence, base pair composition, and base pair sequence of the common arm are demonstrated for the first time by means of proton 500-MHz nuclear magnetic resonance. From Mg2+ titration, temperature variation, ring current calculations, sequence comparisons, and proton homonuclear Overhauser enhancement experiments, additional base pairs in the common arm of the eukaryotic 5S RNA secondary structure are detected. Two base pairs, G41 X C34 and A42 X U33 in the hairpin loop, could account for the lack of binding between the conserved GAAC segment of 5S RNA and the conserved Watson-Crick-complementary GT psi C segment of tRNAs.     

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.     

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.     

Nucleotide sequence of Neurospora crassa cytoplasmic initiator tRNA.

Initiator methionine tRNA from the cytoplasm of Neurospora crassa has been purified and sequenced. The sequence is: pAGCUGCAUm1GGCGCAGCGGAAGCGCM22GCY*GGGCUCAUt6AACCCGGAGm7GU (or D) - CACUCGAUCGm1AAACGAG*UUGCAGCUACCAOH. Similar to initiator tRNAs from the cytoplasm of other eukaryotes, this tRNA also contains the sequence -AUCG- instead of the usual -TphiCG (or A)- found in loop IV of other tRNAs. The sequence of the N. crassa cytoplasmic initiator tRNA is quite different from that of the corresponding mitochondrial initiator tRNA. Comparison of the sequence of N. crassa cytoplasmic initiator tRNA to those of yeast, wheat germ and vertebrate cytoplasmic initiator tRNA indicates that the sequences of the two fungal tRNAs are no more similar to each other than they are to those of other initiator tRNAs.     

Sequence and structure requirements for the biosynthesis of pseudouridine (psi 35) in plant pre-tRNA(Tyr).

All eukaryotic cytoplasmic tRNAs(Tyr) contain pseudouridine in the centre of the anticodon (psi 35). Recently, it has been shown that the formation of psi 35 is dependent on the presence of introns in tRNA(Tyr) genes. Furthermore, we have investigated the structural and sequence requirements for the biosynthesis of psi 35. A number of mutant genes were constructed by oligonucleotide-directed mutagenesis of a cloned Arabidopsis tRNA(Tyr) gene. Nucleotide exchanges were produced in the first and third positions of the anticodon and at positions adjacent to the anticodon. Moreover, insertion and deletion mutations were made in the anticodon stem and in the intron. The mutant genes were transcribed in HeLa cell extract and the pre-tRNAs(Tyr) were used for studying psi 35 biosynthesis in HeLa cell and wheat germ extracts. We have made the following observations about the specificity of plant and vertebrate psi 35 syntheses: (i) insertion or deletion of one base pair in the anticodon stem does not influence the efficiency and accuracy of the psi 35 synthase; (ii) the presence of U35 in a stable double-stranded region prevents its modification to psi 35; and (iii) the consensus sequence U33N34U35A36Pu37 in the anticodon loop is an absolute requirement for psi 35 synthesis. Thus, psi 35 synthases recognize both tRNA tertiary structure and specific sequences surrounding the nucleotide to be modified.     

Nucleotide sequence of initiator tRNA from Mycobacterium smegmatis.

The nucleotide sequence of initiator tRNA from Mycobacterium smegmatis was determined to be pCGCGGGGUGGAGCAGCUCGGDAGCUCGCUGGGCUCAUAACCCAGAGm7GUCG CAGGU psi CGm1AAUCCUGUCCCCGCUACCAOH . The nucleotide sequence of Mycobacterium initiator tRNA was found to be the same as that of Streptomyces initiator tRNA, except that G46 and A57 were replaced by m7G46 and G57 , respectively. The striking feature of Mycobacterium initiator tRNA is the absence of ribothymidine at residue 54, and the presence of 1-methyladenosine at residue 58 which makes the sequence of this tRNA similar to that of eukaryotic initiator tRNA.     

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.     

Enzymatic formation of queuosine and of glycosyl queuosine in yeast tRNAs microinjected into Xenopus laevis oocytes. The effect of the anticodon loop sequence

The eukaryotic tRNA-guanine transglycosylases (queuine insertases) catalyse an exchange of guanine for queuine in position 34, the wobble nucleoside, of tRNAs having a GUN anticodon where N (position 36) stands for A, U, C or G. In tRNAAsp (anticodon QUC) and tRNATyr (anticodon Q psi A) from certain eukaryotic cells, the nucleoside Q-34 is further hypermodified into a glycosylated derivative by tRNA-queuine glycosyltransferase. In order to gain insight into the influence of the nucleosides in position 36, 37 and 38 of an anticodon loop on the potential of a tRNA to become a substrate for the two modifying enzymes, we have constructed several variants of yeast tRNAs in which the normal anticodon has been replaced by one of the synthetic anticodons GUA, GUC, GUG or GUU. In yeast tRNAAsp, the nucleosides 37 (m1G) and 38(C) have also been replaced by an adenosine. These reconstructed chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and tested for their ability to react with the oocyte maturation enzymes. Our results indicate that the nucleosides in positions 36, 37 and 38 influence the efficiencies of conversion of G-34 to Q-34 and of Q-34 to glycosyl Q-34; the importance of their effects are much more pronounced on the glycosylation of Q-34 than on the insertion of queuine. The effect of the nucleoside in position 37 is of particular importance in the case of yeast tRNAAsp: the replacement of the naturally occurring m1G-37 by an unmodified adenosine (as it is in X. laevis tRNAAsp), considerably increases the yield of the glycosylation reaction catalysed by the X. laevis tRNA-queuine glycosyltransferase.