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

Abstract

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...

Nucleotide sequence of a yeast mitochondrial threonine‐tRNA able to decode the C—U—N leucine codons

“…the highest homology (59%) is observed with the mt tRNAp (anticodon U-A-G) fromiV. crussa [2]. On the basis of this resemblance, we favor the hypothesis that yeast mt tRNAThr originates from an ancestral mt leucine-tRNA gene having an anticodon U-A-G and a normal size for the corresponding loop.…”

“…Furthermore, the presence of the U-A-G anticodon in this tRNA explains why the C-U-A codon for leucine is translated into threonine in yeast mitochondria [6], unlike in any other mitochondrial or non-mitochondrial genetic systems. In [2] it was shown from determinations of the nucleotide sequences of 6 mt tRNAs that Neurosporu classa mitochondria show unique features in the codon reading patterns, i.e., mt tRNAs containing an unmodified uridine in the first position of the anticodon are capable of reading all four codons of an unmixed codon family, whereas those containing a modified uridine derivative in the same position are restricted to reading mixed codon families ending in A or G. Our previous sequencing of leucine-, aiginine 1 -, tryptophan-, serine 2-and glycine-mt tRNAs from yeast ([22,23], R. P. M., in preparation) showed that the rules governing the specificity of the codon-anticodon interaction which have been described for N. crassa mitochondria [2] are also operative in yeast mitochondria. Our finding that the uridine residue in the U-A-G anticodon of mt tRNATh is unmodified suggests that this tRNA not only translates the C-U-A codon into threonine, but also the 3 other triplets of the C-U-N leucine family.…”

Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid

“…The anticodon stem in TRL-64 can be drawn with 5-8 bp, depending on whether the distal AU residues are paired, and it lacks all of the expected bases, with the possible exception of U37, which is also found in N.crassa mt tRNA Ala (32). There is no variable arm nor are there the requisite numbers of unpaired nucleotides separating the different stems.…”

Celebrating wobble decoding: Half a century and still much is new

“…47 The unmodified U 34 nucleosides of native mitochondrial alanine, leucine, threonine and valine tRNA species in vivo , and that of a totally unmodified tRNA transcript in vitro were shown to read codons ending in U3 and C3, as well as A3 and G3. 48,49 To understand the forces that maintained and propagated tRNAs' anticodon domain modifications throughout all life, first we will discuss the limited number of examples revealed over many years to have unmodified Us and As at wobble position 34, and the influence of position 32 nucleosides on decoding.

10.1080/15476286.2017.1356562-f0003Figure 3.Modifications present in tRNA's anticodon stem and loop domain (ASL) featured in the text and displayed in their neutral state.

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“…1). 49-57 Wobble codon recognition is at its extreme in these circumstances and has been referred to as ‘superwobble’. 58 An unmodified U in tRNA's wobble position 34 facilitates the use of far fewer tRNAs in organelles than the over 40 cytoplasmic species typically reading the 61 amino acid codes.…”