Structure and function of initiator methionine tRNA from the mitochondria of neurospora crassa

Heckman, Joyce E.; Hecker, Lanny I.; Schwartzbach, Steven D.; Barnett, W. Edgar; Baumstark, Barbara R.; RajBhandary, Uttam L.

doi:10.1016/0092-8674(78)90140-x

Cited by 85 publications

(35 citation statements)

References 43 publications

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“…These were also the criteria used to identify the initiator tRNA species in eukarya such as yeast (25), mammalian cells (26), and fungal mitochondria (27) (28), and Thermoplasma acidophilum (28).…”

Section: Resultsmentioning

confidence: 99%

Importance of the Anticodon Sequence in the Aminoacylation of tRNAs by Methionyl-tRNA Synthetase and by Valyl-tRNA Synthetase in an Archaebacterium

RajBhandary

2001

Journal of Biological Chemistry

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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 (CAU3 CUA and CAU3 GAC) in the anticodon sequence of the initiator tRNA on its recognition by the aminoacyl-tRNA synthetases in vivo. The CAU3 CUA mutant was not aminoacylated to any significant extent in vivo, suggesting the importance of the anticodon in aminoacylation of tRNA by methionyltRNA 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 CAU3 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.The sequence and/or structural determinants in the anticodon and in the acceptor stem of tRNAs play an important role in discrimination among tRNAs by aminoacyl-tRNA synthetases. The crystal structure analysis of several aminoacyl-tRNA synthetase-tRNA complexes from eubacteria and eukarya has provided a substantial amount of information on the molecular details of interactions involving these determinants (1-5). However, little is known either at the biochemical level or at the structural level on interaction between aminoacyl-tRNA synthetases and tRNA in archaea. The results of recent work, spurred by a knowledge of the complete genome sequences of several archaea, have highlighted some interesting and surprising differences between three archaeal aminoacyl-tRNA synthetases and their eubacterial and eukaryal counterparts. First, in contrast to lysyl-tRNA synthetases from eubacteria and eukarya, which in general belong to Class II (5), lysyltRNA synthetase from Methanococcus jannaschii belongs to Class I (6, 7). Second, a single polypeptide of M. jannaschii has the activity (8, 9) of both a cysteinyl-tRNA synthetase and prolyl-tRNA synthetase. Third, the M. jannaschii tyrosyl-tRNA synthetase has a truncated C-terminal region and lacks most of the tRNA anticodon binding region seen in tyro...

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Section: Resultsmentioning

confidence: 99%

Importance of the Anticodon Sequence in the Aminoacylation of tRNAs by Methionyl-tRNA Synthetase and by Valyl-tRNA Synthetase in an Archaebacterium

RajBhandary

2001

Journal of Biological Chemistry

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“…Preliminary results indicated that this mutant strain also overproduces initiation factor IF2 by about It is unlikely that a similar "undermodification" of U to T in yeast mitochondrial initiator tRNA is responsible for initiation with Met-tRNA fMet . While N. crassa mitochondrial initiator tRNA normally contains U in place of T in loop IV (20), the S. cerevisiae mitochondrial initiator tRNA is known to contain T (45). S. cerevisiae has a single gene (TRM2) for tRNA uracil-5 methylase, which encodes both the cytoplasmic and mitochondrial forms of the enzyme (21).…”

Section: Discussionmentioning

confidence: 99%

Initiation of Protein Synthesis in Saccharomyces cerevisiae Mitochondria without Formylation of the Initiator tRNA

Holmes

Appling

et al. 2000

J Bacteriol

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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 (MettRNA 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 fMettRNA 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.

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“…In contrast, it appears that all the RNAs necessary for mitochondrial protein synthesis (ribosomal, transfer, messenger) are made inside the mitochondrion. Although the sizes of mitochondrial DNAs from different sources vary, in all cases the mitochondrial DNA codes for [8][9][10][11][12] proteins, at least two ribosomal RNAs, and several tRNAs (2, 3).A puzzling observation until now has been that the number of different tRNA species present in fungal (4, 5), amphibian (6), and mammalian (7) mitochondria is less than the minimum, 32, needed to read all the codons of the genetic code using the codon/anticodon pairing rules proposed by Crick (8). How then does the mitochondrial protein-synthesizing system read all the codons of the genetic code?…”

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confidence: 99%

Novel features in the genetic code and codon reading patterns in Neurospora crassa mitochondria based on sequences of six mitochondrial tRNAs

Heckman

Sarnoff

Alzner-Deweerd

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

241

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We report the sequences of Neurospora crassa mitochondrial alanine, leucinel, leucine2, threonine, tryptophan, and valine tRNAs. On the basis of the anticodon sequences of these tRNAs and of a glutamine tRNA, whose sequence analysis is nearly complete, we infer the following: (i) The N. crassa mitochondrial tRNA species for alanine, leucine2, threonine, and valine, amino acids that belong to four-codon families (GCN, CUN, ACN, and GUN, respectively; N = U, C, A, or G) all contain an unmodified U in the first position of the anticodon. In contrast, tRNA species for glutamine, leucinel, and tryptophan, amino acids that use codons ending in purines (CAt, URJC, and UGG, respectively) contain a modified U derivative in the same position. These findings and the fact that we have not detected any other isoacceptor tRNAs for these amino acids suggest that N. crassa mitochondrial tRNAs containing U in the first position of the anticodon are capable of reading all four codons of a four-codon family whereas those containing a modified U are restricted to reading codons ending in A or G. Such an -expanded codon-reading ability of certain mitochondrial tRNAs will explain how the mitochondrial protein-synthesizing system operates with a much lower number of tRNA species than do systems present in prokaryotes or in eukaryotic cytoplasm. (ii) The anticodon sequence of the N. crassa mitochondrial tryptophan tRNA is U*CA and not CCA or CmCA as is the case with tryptophan tRNAs from prokaryotes or from eukaryotic cytoplasm. Because a tRNA with U*CA in the anticodon would be expected to read the codon UGA, as well as the normal tryptophan codon UGG, this suggests that in N. crassa mitochondria, as in yeast and in human mitochondria, UGA is a codon for tryptophan and not a signal for chain termination. (iii) The anticodon sequences of the two leucine tRNAs indicate that N. crassa mitochondria use both families of leucine codons (UUA and CUN; N = U, C, A, or G) for leucine, in contrast to yeast mitochondria [Li, M. & Tzagoloff, A. (1979) Cell 18, 47-53] in which the CUA leucine codon and possibly the entire CUN family of leucine codons may be translated as threonine.Mitochondria exist within the cytoplasm of eukaryotic cells (1). They contain a DNA genome and a protein-synthesizing system that is distinct from the system in the cytoplasm (2, 3). Virtually all of the protein components of the mitochondrial protein biosynthetic machinery are coded for by the nuclear DNA, made in the cytoplasm, and imported into the mitochondria. In contrast, it appears that all the RNAs necessary for mitochondrial protein synthesis (ribosomal, transfer, messenger) are made inside the mitochondrion. Although the sizes of mitochondrial DNAs from different sources vary, in all cases the mitochondrial DNA codes for [8][9][10][11][12] proteins, at least two ribosomal RNAs, and several tRNAs (2, 3).A puzzling observation until now has been that the number of different tRNA species present in fungal (4, 5), amphibian (6), and mammalian (7) mitocho...

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Structure and function of initiator methionine tRNA from the mitochondria of neurospora crassa

Cited by 85 publications

References 43 publications

Importance of the Anticodon Sequence in the Aminoacylation of tRNAs by Methionyl-tRNA Synthetase and by Valyl-tRNA Synthetase in an Archaebacterium

Importance of the Anticodon Sequence in the Aminoacylation of tRNAs by Methionyl-tRNA Synthetase and by Valyl-tRNA Synthetase in an Archaebacterium

Initiation of Protein Synthesis in Saccharomyces cerevisiae Mitochondria without Formylation of the Initiator tRNA

Novel features in the genetic code and codon reading patterns in Neurospora crassa mitochondria based on sequences of six mitochondrial tRNAs

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