Search results for "trna"

Iron in Translation: From the Beginning to the End

2021

Iron is an essential element for all eukaryotes, since it acts as a cofactor for many enzymes involved in basic cellular functions, including translation. While the mammalian iron-regulatory protein/iron-responsive element (IRP/IRE) system arose as one of the first examples of translational regulation in higher eukaryotes, little is known about the contribution of iron itself to the different stages of eukaryotic translation. In the yeast Saccharomyces cerevisiae, iron deficiency provokes a global impairment of translation at the initiation step, which is mediated by the Gcn2-eIF2α pathway, while the post-transcriptional regulator Cth2 specifically represses the translation of a subgroup of…

Mapping the tRNA binding site on the surface of human DNMT2 methyltransferase.

2012

The DNMT2 enzyme methylates tRNA-Asp at position C38. Because there is no tRNA–Dnmt2 cocrystal structure available, we have mapped the tRNA binding site of DNMT2 by systematically mutating surface-exposed lysine and arginine residues to alanine and studying the tRNA methylation activity and binding of the corresponding variants. After mutating 20 lysine and arginine residues, we identified eight of them that caused large (>4-fold) decreases in catalytic activity. These residues cluster within and next to a surface cleft in the protein, which is large enough to accommodate the tRNA anticodon loop and stem. This cleft is located next to the binding pocket for the cofactor S-adenosyl-l-methion…

Single-Molecule FRET Reveals a Cooperative Effect of Two Methyl Group Modifications in the Folding of Human Mitochondrial tRNALys

2011

Summary Using a combination of advanced RNA synthesis techniques and single molecule spectroscopy, the deconvolution of individual contributions of posttranscriptional modifications to the overall folding and stabilization of human mitochondrial tRNA Lys is described. An unexpected destabilizing effect of two pseudouridines on the native tRNA folding was evidenced. Furthermore, the presence of m 2 G10 alone does not facilitate the folding of tRNA Lys , but a stabilization of the biologically functional cloverleaf shape in conjunction with the principal stabilizing component m 1 A9 exceeds the contribution of m 1 A alone. This constitutes an unprecedented cooperative effect of two nucleotide…

RNA nucleotide methylation

2011

Methylation of RNA occurs at a variety of atoms, nucleotides, sequences and tertiary structures. Strongly related to other posttranscriptional modifications, methylation of different RNA species includes tRNA, rRNA, mRNA, tmRNA, snRNA, snoRNA, miRNA, and viral RNA. Different catalytic strategies are employed for RNA methylation by a variety of RNA-methyltransferases which fall into four superfamilies. This review outlines the different functions of methyl groups in RNA, including biophysical, biochemical and metabolic stabilization of RNA, quality control, resistance to antibiotics, mRNA reading frame maintenance, deciphering of normal and altered genetic code, selenocysteine incorporation,…

Structural insights into the GTPase domain of Escherichia coli MnmE protein

2007

The Escherichia coli MnmE protein is a 50-kDa multidomain GTPase involved in tRNA modification. Its homologues in eukaryotes are crucial for mitochondrial respiration and, thus, it is thought that the human protein might be involved in mitochondrial diseases. Unlike Ras, MnmE shows a high intrinsic GTPase activity and requires effective GTP hydrolysis, and not simply GTP binding, to be functionally active. The isolated MnmE G-domain (165 residues) conserves the GTPase activity of the entire protein, suggesting that it contains the catalytic residues for GTP hydrolysis. To explore the GTP hydrolysis mechanism of MnmE, we analyzed the effect of low pH on binding and hydrolysis of GTP, as well…

Small genomes and the difficulty to define minimal translation and metabolic machineries

2015

The notion of minimal life has sparked the interest of scientists in different fields, ranging from the origin-of-life research to biotechnology-oriented synthetic biology. Whether the interest is focused on the emergence of protocells out of prebiotic systems or the design of a cell chassis ready to incorporate new devices and functions, proposing minimal combinations of genes for life is not a trivial task. Using comparative genomics and biochemistry of endosymbionts (i.e., intracellular mutualistic symbionts) and intracellular parasites, a decade ago we proposed the core of a minimal gene set for a simple heterotrophic cell adapted to a chemically complex environment. In this work, we di…

Dynamic modulation of Dnmt2-dependent tRNA methylation by the micronutrient queuine

2015

Dnmt2 enzymes are cytosine-5 methyltransferases that methylate C38 of several tRNAs. We report here that the activities of two Dnmt2 homologs, Pmt1 from Schizosaccharomyces pombe and DnmA from Dictyostelium discoideum, are strongly stimulated by prior queuosine (Q) modification of the substrate tRNA. In vivo tRNA methylation levels were stimulated by growth of cells in queuine-containing medium; in vitro Pmt1 activity was enhanced on Q-containing RNA; and queuine-stimulated in vivo methylation was abrogated by the absence of the enzyme that inserts queuine into tRNA, eukaryotic tRNA-guanine transglycosylase. Global analysis of tRNA methylation in S. pombe showed a striking selectivity of Pm…

The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu

2014

Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice. Unexpectedly, it had only a weak activity toward its matching Geobacter tRNA-Asp, but methylated Geobacter tRNA-Glu with good activity. In agreement with this result, we show that tRNA-Glu is methylated in Geobacter while the methylation is absent in tRNA-Asp. The activities of Dnmt2 enzymes from Homo sapiens, Drosophila melanogaster, Schizosaccharomyces pombe and Dictyostelium discoideum for methylation of the …

2020

Abstract RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2′-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS s…

Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling.

2010

This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNA(Phe). LC/MS and LC/MS/MS techniques were used to detect and characterize the modified nucleoside as well as its cycloaddition product with a fluorescent azide. The latter resulted from a labeling reaction via Cu(I…