Structure-function analysis of Escherichia coli MnmG (GidA), a highly conserved tRNA-modifying enzyme

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-A resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.This work was supported by grant GSP-48370 from the Canadian Institutes of Health Research (to M.C. and A.M.) and grant BFU2007-66509 from the Ministerio de Ciencia e Innovación (to M.-E.A.).Peer reviewe

Further insights into the tRNA modification process controlled by proteins MnmE and GidA of Escherichia coli

In Escherichia coli, proteins GidA and MnmE are involved in the addition of the carboxymethylaminomethyl (cmnm) group onto uridine 34 (U34) of tRNAs decoding two-family box triplets. However, their precise role in the modification reaction remains undetermined. Here, we show that GidA is an FAD-binding protein and that mutagenesis of the N-terminal dinucleotide-binding motif of GidA, impairs capability of this protein to bind FAD and modify tRNA, resulting in defective cell growth. Thus, GidA may catalyse an FAD-dependent reaction that is required for production of cmnmU34. We also show that GidA and MnmE have identical cell location and that both proteins physically interact. Gel filtration and native PAGE experiments indicate that GidA, like MnmE, dimerizes and that GidA and MnmE directly assemble in an α2β2 heterotetrameric complex. Interestingly, high-performance liquid chromatography (HPLC) analysis shows that identical levels of the same undermodified form of U34 are present in tRNA hydrolysates from loss-of-function gidA and mnmE mutants. Moreover, these mutants exhibit similar phenotypic traits. Altogether, these results do not support previous proposals that activity of MnmE precedes that of GidA; rather, our data suggest that MnmE and GidA form a functional complex in which both proteins are interdependent

Structural Basis for Fe–S Cluster Assembly and tRNA Thiolation Mediated by IscS Protein–Protein Interactions

Crystal structures reveal how distinct sites on the cysteine desulfurase IscS bind two different sulfur-acceptor proteins, IscU and TusA, to transfer sulfur atoms for iron-sulfur cluster biosynthesis and tRNA thiolation

Effect of the genetic background and RNAi silencing on <i>C</i>. <i>elegans</i> lifespan at 20°C.

<p>Effect of the genetic background and RNAi silencing on <i>C</i>. <i>elegans</i> lifespan at 20°C.</p

Expression of UPR^mt markers in <i>mttu-1</i>, <i>mtcu-</i>1 and <i>mtcu-2</i> mutant strains.

<p><b>(A and B)</b> Expression of the <i>hsp-6</i>_<i>p</i>::GFP or <i>hsp-60</i>_<i>p</i>::GFP reporters in the indicated strains. Representative fluorescence micrographs of wild-type and single mutants harbouring <i>hsp-6</i>_<i>p</i>::GFP or <i>hsp-60</i>_<i>p</i>::GFP transgenes at L4 stage of development are shown in (A). Quantification is shown in (B) (n>20). * denotes p<0.05, **, p<0.01 and ***, p<0.001. <b>(C)</b> Fluorescence micrographs of wild-type and single mutants harbouring <i>hsp-6</i>_<i>p</i>::GFP or <i>hsp-60</i>_<i>p</i>::GFP transgenes at L4 stage of development after <i>cyc-1</i>(RNAi) from the L1 stage. <b>(D)</b> Fluorescence micrographs of one-day old, adult wild type and single mutants harbouring <i>hsp-6</i>_<i>p</i>::GFP transgene exposed to 1 mM paraquat for 24 h. Note that <i>cyc-1</i>(RNAi) (C) and paraquat treatment (D) cause marked increases in GFP expression. <b>(E)</b> Quantitation of <i>clpp-1</i> and <i>ubl-5</i> mRNA levels by qRT-PCR in the indicated strains at L4 stage (n≥3). The mRNA levels were normalized to those of <i>act-1</i> in the wild-type strain. Statistical significance was evaluated with Student’s unpaired t-test. Error bars indicate standard deviation (SD). *, p<0.05.</p

Modification of the wobble uridine (U₃₄) in mitochondrial and bacterial tRNAs.

<p>Schema of the U₃₄ modification pathways in human and yeast mt-tRNAs and <i>Escherichia coli</i> tRNAs <b>(A)</b> and <i>A</i>. <i>suum</i> mt-tRNAs <b>(B)</b>. In A, proteins GTPBP3, MTO1, and TRMU (also named MTU1) from humans, and MSS1, MTO1, and MTU1, from yeast, are orthologous of the bacterial MnmE, MnmG and MnmA proteins, respectively. Taurine (humans) and glycine (<i>E</i>. <i>coli</i> and yeast) are used to introduce the τm and cmnm groups into position 5 of U₃₄. In B, MTCU-1, MTCU-2 and MTTU-1 are the nematode orthologues of GTPBP3, MTO1, and TRMU, respectively, and their roles are inferred from studies of the <i>E</i>. <i>coli</i> and yeast proteins. The fractions of <i>A</i>. <i>suum</i> modified mt-tRNAs, according to the study by Sakurai <i>et al</i>. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006921#pgen.1006921.ref028" target="_blank">28</a>], are indicated below the schema. Note that mt-tRNA^Glu, mt-tRNA^Lys and mt-tRNA^Gln are fully modified at position 5 but lack thiolation at position 2, whereas most (~90%) is modified at both positions (2 and 5), and about 50% of mt-tRNA^Trp molecules do not contain any modification at the U₃₄.</p

Simultaneous inactivation of MTTU-1 and MTCU-2 leads to lifespan extension in <i>C</i>. <i>elegans</i>.

<p><b>(A)</b> Survival of the wild-type strain and the <i>mttu-1</i>, <i>mtcu-1</i> and <i>mtcu-2</i> single mutants at 20°C (n = 4). <b>(B)</b> Survival of the wild-type strain and the <i>mtcu-2; mttu-1</i> double mutant at 20°C (n = 3). <b>(C-J)</b> <i>aak-1</i> (n = 2) (C), <i>aak-2</i> (n = 2) (D), <i>daf-2</i> (n = 1) (E), <i>rict-1</i> (n = 1) (F), <i>daf-16</i> (n = 2) (G), <i>kri-1</i> (n = 2) (H), <i>daf-9</i> (n = 2) (I), and <i>daf-12</i> (n = 2) (J) silencing effect on the survival of the N2 and <i>mtcu-2; mttu-1</i> strains at 20°C. The empty vector L4440 was used as a negative control. Animals used for controls were of the same age as the experimental animals. To avoid disturbing embryonic development, silencing was started at the L4 stage (pointed with and arrow and a dashed line). Statistical significance was evaluated with Log-rank (Mantel Cox test) and Gehan-Breslow-Wilcoxon test and statistics are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006921#pgen.1006921.t001" target="_blank">Table 1</a>.</p

Simultaneous lack of mitochondrial MTTU-1 and MTCU-2 proteins is associated with embryonic lethality, developmental defects and sterility.

<p><b>(A)</b> Silencing of <i>mttu-1</i> in the <i>mtcu-2</i> mutant (from L4 stage onwards) at 25°C produces a slower rate of development and sterility of their progeny. <b>(B)</b> and <b>(C)</b> Silencing of <i>mtcu-2</i> in the <i>mttu-1</i> mutant (from L4 stage onwards) at 25°C causes arrest of development at the L1-L2 stages (B) and embryonic lethality (C). The total number of eggs and the number that failed to hatch were quantified (n≥5). **p<0.01, ***p<0.001. Statistical significance was evaluated with Student’s unpaired t-test. Error bars indicate standard deviation (SD).</p