Single-Molecule FRET Reveals a Cooperative Effect of Two Methyl Group Modifications in the Folding of Human Mitochondrial tRNALys (original) (raw)
Related papers
Biochemistry, 1999
We have previously shown by chemical and enzymatic structure probing that, opposite to the native human mitochondrial tRNA Lys , the corresponding in vitro transcript does not fold into the expected tRNA-specific cloverleaf structure. This RNA folds into a bulged hairpin, including an extended amino acid acceptor stem, an extra large loop instead of the T-stem and loop, and an anticodon-like domain. Hence, one or several of the six modified nucleotides present in the native tRNA are required and responsible for its cloverleaf structure. Phylogenetic comparisons as well as structural analysis of variant transcripts had pointed to m 1 A9 as the most likely important modified nucleotide in the folding process.
Nucleic Acids Research, 1998
Direct sequencing of human mitochondrial tRNA Lys shows the absence of editing and the occurrence of six modified nucleotides (m 1 A9, m 2 G10, Ψ27, Ψ28 and hypermodified nucleotides at positions U34 and A37). This tRNA folds into the expected cloverleaf, as confirmed by structural probing with nucleases. The solution structure of the corresponding in vitro transcript unexpectedly does not fold into a cloverleaf but into an extended bulged hairpin. This non-canonical fold, established according to the reactivity to a large set of chemical and enzymatic probes, includes a 10 bp aminoacyl acceptor stem (the canonical 7 bp and 3 new pairs between residues 8-10 and 65-63), a 13 nt large loop and an anticodon-like domain. It is concluded that modified nucleotides have a predominant role in canonical folding of human mitochondrial tRNA Lys . Phylogenetic comparisons as well as structural probing of selected in vitro transcribed variants argue in favor of a major contribution of m 1 A9 in this process.
The tRNA-Induced Conformational Activation of Human Mitochondrial Phenylalanyl-tRNA Synthetase
Structure, 2008
All class II aminoacyl-tRNA synthetases (aaRSs) are known to be active as functional homodimers, homotetramers, or heterotetramers. However, multimeric organization is not a prerequisite for phenylalanylation activity, as monomeric mitochondrial phenylalanyl-tRNA synthetase (PheRS) is also active. We herein report the structure, at 2.2 Å resolution, of a human monomeric mitPheRS complexed with Phe-AMP. The smallest known aaRS, which is, in fact, 1/5 of a cytoplasmic analog, is a chimera of the catalytic module of the a and anticodon binding domain (ABD) of the bacterial b subunit of (ab) 2 PheRS. We demonstrate that the ABD located at the C terminus of mitPheRS overlaps with the acceptor stem of phenylalanine transfer RNA (tRNA Phe) if the substrate is positioned in a manner similar to that seen in the binary Thermus thermophilus complex. Thus, formation of the PheRS-tRNA Phe complex in human mitochondria must be accompanied by considerable rearrangement (hinge-type rotation through $160) of the ABD upon tRNA binding.
A Noncanonical Tertiary Conformation of a Human Mitochondrial Transfer RNA
Biochemistry, 1995
Transfer RNAs possess highly conserved secondary structures, and crystallographic studies suggest a common, L-shaped tertiary conformation in which the anticodon and acceptor stems are disposed at approximately right angles to one another. However, many animal mitochondrial tRNAs possess unusual secondary structures, and little is known regarding their tertiary conformations, in particular, the relative orientations of their acceptor and anticodon stems. To address this issue, we have constructed heteroduplex RNA molecules corresponding to human mitochondrial and cytoplasmic lysyl tRNAs in which the acceptor and anticodon stems of each tRNA have been extended by approximately 70 base pairs. The rotational decay times of the two "extended" tRNALyS species were compared to the decay times of a linear RNA control and to an extended yeast cytoplasmic tRNAPhe species whose interstem angle had been reported previously. Whereas the apparent interstem angle of the human cytoplasmic tRNALyS species is essentially identical to that of the yeast tRNAPhe heteroduplex, with both conforming to the canonical L-shape, the angle for the mitochondrial tRNALys construct is much larger (-140"). Thus, the universal L-shape may not be applicable to noncanonical mitochondrial tRNAs, a finding of significance for both tRNA evolution and mitochondrial disease.
The Journal of Biochemistry, 2020
A fundamental aspect of mitochondria is that they possess DNA and protein translation machinery. Mitochondrial DNA encodes 22 tRNAs that translate mitochondrial mRNAs to 13 polypeptides of respiratory complexes. Various chemical modifications have been identified in mitochondrial tRNAs via complex enzymatic processes. A growing body of evidence has demonstrated that these modifications are essential for translation by regulating tRNA stability, structure and mRNA binding, and can be dynamically regulated by the metabolic environment. Importantly, the hypomodification of mitochondrial tRNA due to pathogenic mutations in mitochondrial tRNA genes or nuclear genes encoding modifying enzymes can result in life-threatening mitochondrial diseases in humans. Thus, the mitochondrial tRNA modification is a fundamental mechanism underlying the tight regulation of mitochondrial translation and is essential for life. In this review, we focus on recent findings on the physiological roles of 5-tau...
Nature Communications
Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m3C32 in the human mitochondrial (mt-)tRNAThr and mt-tRNASer(UCN). METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mt-tRNA recognition elements revealed U34G35 and t6A37/(ms2)i6A37, present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C32. Several lines of evidence demonstrate the influence of U34, G35, and the m3C32 and t6A37/(ms2)i6A37 modifications in mt-tRNAThr/Ser(UCN) on the structure of these mt-tRNAs. Although mt-tRNAThr/Ser(UCN) lacking METTL8-mediated m3C32 are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack...
Journal of Biological …, 2008
3 The abbreviations used are: EF-Tu mt , mitochondrial elongation factor Tu; hmtRNA Met , human mitochondrial tRNA Met ; MetRS, methionyl-tRNA synthetase; hmMetRS, human mitochondrial methionyl-tRNA synthetase; MTF, methionyl-tRNA transformylase; SHAPE, selective 2Ј-hydroxyl acylation analyzed by primer extension; PMSF, phenylmethylsulfonyl fluoride; ME, -mercaptoethanol; 1M7, 1-methyl-7-nitroisatoic anhydride.
Aminoacylation properties of pathology-related human mitochondrial tRNALys variants
RNA, 2004
In vitro transcription has proven to be a successful tool for preparation of functional RNAs, especially in the tRNA field, in which, despite the absence of post-transcriptional modifications, transcripts are correctly folded and functionally active. Human mitochondrial (mt) tRNA Lys deviates from this principle and folds into various inactive conformations, due to the absence of the post-transcriptional modification m 1 A9 which hinders base-pairing with U64 in the native tRNA. Unavailability of a functional transcript is a serious drawback for structure/function investigations as well as in deciphering the molecular mechanisms by which point mutations in the mt tRNA Lys gene cause severe human disorders. Here, we show that an engineered in vitro transcribed "pseudo-WT" tRNA Lys variant is efficiently recognized by lysyl-tRNA synthetase and can substitute for the WT tRNA as a valuable reference molecule. This has been exploited in a systematic analysis of the effects on aminoacylation of nine pathology-related mutations described so far. The sole mutation located in a loop of the tRNA secondary structure, A8344G, does not affect aminoacylation efficiency. Out of eight mutations located in helical domains converting canonical Watson-Crick pairs into G-U pairs or C•A mismatches, six have no effect on aminoacylation (A8296G, U8316C, G8342A, U8356C, U8362G, G8363A), and two lead to drastic decreases (5000-to 7000-fold) in lysylation efficiencies (G8313A and G8328A). This screening, allowing for analysis of the primary impact level of all mutations affecting one tRNA under comparable conditions, indicates distinct molecular origins for different disorders.