The turnover of methionine in the Met-tRNA pool and the control of protein synthesis in reticulocyte lysates: no evidence that haem-deficiency promotes deacylation of Met-tRNAf.
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Animals↗
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Results for “RNA, Transfer, Met”
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Nucleotide sequence of Chlamydomonas reinhardtii mitochondrial genes coding for tRNA(Gln) (UUG) and tRNA(Met) (CAU).
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Base Sequence↗
Function of eukaryotic translation initiation factor 1A (eIF1A) (formerly called eIF-4C) in initiation of protein synthesis.
We have used an efficient in vitro translation initiation system to show that the mammalian 17-kDa eukaryotic initiation factor, eIF1A (formerly designated eIF-4C), is essential for transfer of the initiator Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA to form the 40 S preinitiation complex (40 S.Met-tRNAf.eIF2.GTP). Furthermore, eIF1A acted catalytically in this reaction to mediate highly efficient transfer of the Met-tRNAf.eIF2.GTP ternary complex to 40 S ribosomal subunits. The 40 S complex formed was free of eIF1A indicating that its role in 40 S preinitiation complex formation is not to stabilize the binding of Met-tRNAf to 40 S ribosomes. Additionally, the eIF1A-mediated 40 S initiation complex formed in the presence of AUG codon efficiently joined 60 S ribosomal subunits in an eIF5-dependent reaction to form a functional 80 S initiation complex. In contrast to other reports, we found that eIF1A plays no role either in the subunit joining reaction or in the generation of ribosomal subunits from 80 S ribosomes. Our results indicate that the major function of eIF1A is to mediate the transfer of Met-tRNAf to 40 S ribosomal subunits to form the 40 S preinitiation complex.
Animals↗
Characterization of the C2 subdomain of yeast mitochondrial initiation factor 2.
The COOH-terminal part of the yeast mitochondrial initiation factor 2 (ymIF2), containing the C2 subdomain, was expressed and purified as a histidine-tagged polypeptide of 137 amino acids. Like the recombinant full-length protein, the C2 subdomain binds both formyl-Met-tRNA(f)(Met) and unformylated Met-tRNA(f)(Met) with only a small preference for the former species. Formation of a binary complex between the C2 subdomain or the full-length ymIF2 and initiator tRNA was also assessed by fluorescence measurements. The binding of coumarin-Met-tRNA(f) to either protein caused a blue shift of the coumarin emission spectrum and an increase in anisotropy. Full-length ymIF2 is functionally competent in forming an initiation complex and supporting formation of the first peptide bond on Escherichia coli ribosomes. The results demonstrate that ymIF2 has the same domain structure and biochemical properties of a typical IF2 species as found in bacteria or mammalian mitochondria--but with enhanced ability to bind unformylated initiator Met-tRNA.
Coumarins↗
Studies on the mechanism of action of a eukaryotic codon-dependent factor specific for initiator Met-tRNAf and ribosomal 40 S subunits.
A putative eukaryotic initiation factor purified from rat liver cytosol promotes the ApUpGp- and Mg2+-dependent, GTP-independent binding of initiator Met-tRNAf to ribosomal 40 S subunits. The isotopically-labeled factor binds specifically to 40 S subunits to form a binary complex which can then bind Met-tRNAf in the presence of ApUpGp. When 60 S subunits are added, an 80 S complex is formed which contains Met-tRNAf but not the factor. The Met-tRNAf bound to 80 S ribosomes reacts quantitatively with puromycin. Purified 40 S subunits exist in the form of monomers and dimers. The factor converts monomers, which appear to contain one molecule of tRNA, to a form that does not contain tRNA and dimerizes readily. Deacylated tRNA inhibits dimerization and, in the presence of the factor, converts dimers to the monomeric form; thus, the factor appears to act in a reversible manner. This protein factor may play a role in the removal of tRNA from 40 S subunits, which could be generated as a consequence of chain termination, a prerequisite to the bindig of Met-tRNAf.
Animals↗
Vaccinia viral core inhibits Met-tRNA.40S invitation complex formation with physiological mRNAs.
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Animals↗
The primary structure of cytoplasmic initiator tRNA(Met) from Schizosaccharomyces pombe.
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Base Sequence↗
Yeast tRNA(Met) recognition by methionyl-tRNA synthetase requires determinants from the primary, secondary and tertiary structure: a review.
The primordial role of the CAU anticodon in methionine identity of the tRNA has been established by others nearly a decade ago in Escherichia coli and yeast tRNA(Met). We show here that the CAU triplet alone is unable to confer methionine acceptance to a tRNA. This requires the contribution of the discriminatory base A73 and the non-anticodon bases of the anticodon loop. To better understand the functional communication between the anticodon and the active site, we analysed the binding and aminoacylation of tRNA(Met) based anticodon and acceptor-stem minihelices and of tRNA(Met) chimeras where the central core region of yeast tRNA(Met) is replaced by that of unusual mitochondrial forms lacking either a D-stem or a T-stem. These studies suggest that the high selectivity of the anticodon bases in tRNA(Met) implies the L-conformation of the tRNA and the presence of a D-stem. The importance of a L-structure for recognition of tRNA(Met) was also deduced from mutations of tertiary interactions known to play a general role in tRNA(Met) folding.
Amino Acid Sequence↗
The ability of bovine mitochondrial transfer RNAMet to decode AUG and AUA codons.
The ability of bovine mitochondrial tRNA(Met) with the anticodon f5CAU (where f5C is 5-formylcytidine) to decode AUG and AUA codons was examined in a codon-dependent ribosomal binding assay. The AUG codon stimulated the binding of Met-tRNA(Met) to mitochondrial ribosomes in the presence of EF-Tu/TSmt. In contrast, the AUA codon did not promote the binding to mitochondrial Met-tRNA to the ribosome. To investigate the translation of the AUG and AUA codons more fully, an in vitro translation system from bovine liver mitochondria was developed. The activity of this system was greatly enhanced by the addition of 1 mM spermine and reached about half the activity observed with a comparable translational system from E coli. Two types of mRNA containing either AUG or AUA codons were synthesized using T7 RNA polymerase to transcribe their chemically synthesized genes. In the E coli system, the AUG-containing mRNA was translated as Met and the AUA-containing mRNA was translated as Ile. The AUG-containing mRNA but not the AUA-containing mRNA was translated as Met by the mitochondrial translational system. The process by which the AUA codon is translated as Met in the mitochondrial system remains to be clarified.
Animals↗
Energy cost of proofreading in vivo: the charging of methionine tRNAs in Escherichia coli.
Previous in vitro work has shown that Escherichia coli methionyl-tRNA synthetase has a limited ability to discriminate against cognate methionine in the editing site designed for noncognate homocysteine. As a result, a small fraction of the correct product Met-tRNA is deacylated with the formation of a cyclic sulfonium compound, S-methyl-homocysteine thiolactone. This is exploited here to estimate energy costs associated with the destruction of a correct product by methionyl-tRNA synthetase in bacterial cells. In vivo measurements of S-methyl-homocysteine thiolactone indicate that in Escherichia coli 3.3 molecules of Met-tRNA are destroyed by deacylation per 10,000 molecules of Met-tRNA successfully transferring methionine to protein. This number of destroyed molecules of a correct product, Met-tRNA, is 30 times lower than the number of destroyed molecules of an incorrect product, homocysteinyl adenylate. Thus, most of the energy cost of proofreading in vivo is due to editing of the noncognate amino acid.
Energy Metabolism↗
Equilibration of methionine into the Met-tRNA pool of rabbit reticulocyte lysates. Additional evidence that deacylation of ribosomal subunit-bound Met-tRNAf does occur in heme-deficiency.
We have measured the rate of equilibration of [35S]methionine into the Met-tRNA pool of rabbit reticulocyte lysate as in [FEBS Lett. (1982) 143, 301-305]. Our results indicate that hemin-deficiency inhibits the equilibration of methionine into the tRNA pool much less than protein synthesis or the equilibration of alanine into the tRNA pool, whereas cycloheximide inhibits these processes similarly. This finding is consistent with our previous data and supports the hypothesis that with hemin-deficiency much of the Met-tRNAf that becomes bound to 40 S subunits subsequently undergoes enzymatic deacylation.
Animals↗
Interaction between non-formylated initiator Met-tRNA(fMet) and the ribosomal A-site from Escherichia coli.
We report studies in vitro of the interaction between non-formylated initiator Met-tRNA(fMet) and 70S ribosomes. The binding of Met-tRNA(fMet) to ribosomes carrying fMet-tRNA(fMet) in the P-site is strongly stimulated by elongation factor EF-Tu:GTP in the presence of (AUG)3. The enzymatically bound Met-tRNA(fMet) does not react with puromycin. The bound Met-tRNA(fMet) can accept formylmethionine from P-site-bound fMet-tRNA(fMet). These results demonstrate a functionally active binding at the ribosomal A-site. Partial ribonuclease digestion (footprinting) was used to study the sites in Met-tRNA(fMet) which are involved in the interaction with the ribosomal A-site. The results show that a large part of the tRNA molecule is protected by the ribosome against ribonuclease digestion. In addition to the protection found in the amino acid region and the anticodon arm, protection is seen in the D-loop and in the extra arm. No region within the bound tRNA is found to be more accessible for RNases than in the free Met-tRNA(fMet). The reported enhancement of ribonuclease cuts in the D- and T-arms of A-site-bound Phe-tRNAPhe is thus not found in A-site bound Met-tRNA(fMet).
Base Sequence↗
Use of a dot blot hybridization method for identification of pure tRNA species on different membranes.
The characterization of a tRNA in purification procedures usually involves aminoacylation assays but recently, the hybridization by dot blot with specific oligonucleotides as probes has been used for the tRNA identification. We present here an optimization of a dot blot hybridization method for the tRNA detection by comparing the efficiency of eight different nylon membranes. Neutral 0.22 microns porosity membranes (Nytran, Biodine A) give the best detection efficiency when small quantities of material (less than 40 ng of tRNA) are dotted on filter; by contrast, neutral 0.45 microns porosity membranes (such as Hybond N) are the most efficient when larger quantities of tRNA are dotted on the filter. The described technique allows to detect less than 20 pg of a pure tRNA species. Its use in the identification of Saccharomyces cerevisiae initiator tRNA(Met) in counter-current distribution fractions is shown.
Autoradiography↗
Binding of the anticodon domain of tRNA(fMet) to Escherichia coli methionyl-tRNA synthetase.
A stem and loop RNA domain carrying the methionine anticodon (CAU) was designed from the tRNA(fMet) sequence and produced in vitro. This domain makes a complex with methionyl-tRNA synthetase (Kd = 38(+/- 5) microM; 25 degrees C, pH 7.6, 7 mM-MgCl2). The formation of this complex is dependent on the presence of the cognate CAU anticodon sequence. Recognition of this RNA domain is abolished by a methionyl-tRNA synthetase mutation known to alter the binding of tRNA(Met).
Anticodon↗
Structural basis for anticodon recognition by methionyl-tRNA synthetase.
In the 2.7-A resolution crystal structure of methionyl-tRNA synthetase (MetRS) in complex with tRNA(Met) and a methionyl-adenylate analog, the tRNA anticodon loop is distorted to form a triple-base stack comprising C34, A35 and A38. A tryptophan residue stacks on C34 to extend the triple-base stack. In addition, C34 forms Watson-Crick-type hydrogen bonds with Arg357. This structure resolves the longstanding question of how MetRS specifically recognizes tRNA(Met).
Amino Acid Sequence↗
Trinucleotide repeat polymorphism at the human met-tRNA-i gene 1 (TRMI).
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Base Sequence↗
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).
Animals↗
Effect of L-methioninyl adenylate on the level of aminoacylation in vivo of tRNA(Met) from Escherichia coli K12.
In cells of E.coli K12 grown exponentially in minimal medium, tRNA(met), tRNA(leu) and tRNA(ile) are aminoacylated at 100%, 80% and 64%, respectively. On addition of L-methioninyl adenylate to the growth medium, one observes a specific deacylation of tRNA(met). When more than 35% of tRNA(met) is deacylated, growth rate is reduced and becomes proportional to the amount of methionyl-tRNA formed.
Adenosine Monophosphate↗