The GTPase Activity and C-terminal Cysteine of the Escherichia coli MnmE Protein Are Essential for Its tRNA Modifying Function

Abstract

The Escherichia coli MnmE protein is a three-domain protein that exhibits a very high intrinsic GTPase activity and low affinity for GTP and GDP. The middle GTPase domain, when isolated, conserves the high intrinsic GTPase activity of the entire protein, and the C-terminal domain contains the only cysteine residue present in the molecule. MnmE is an evolutionarily conserved protein that, in E. coli, has been shown to control the modification of the uridine at the wobble position of certain tRNAs. Here we examine the biochemical and functional consequences of altering amino acid residues within conserved motifs of the GTPase and C-terminal domains of MnmE. Our results indicate that both domains are essential for the MnmE tRNA modifying function, which requires effective hydrolysis of GTP. Thus, it is shown for the first time that a confirmed defect in the GTP hydrolase activity of MnmE results in the lack of its tRNA modifying function. Moreover, the mutational analysis of the GTPase domain indicates that MnmE is closer to classical GTPases than to GTP-specific metabolic enzymes. Therefore, we propose that MnmE uses a conformational change associated with GTP hydrolysis to promote the tRNA modification reaction, in which the C-terminal Cys may function as a catalytic residue. We demonstrate that point mutations abolishing the tRNA modifying function of MnmE confer synthetic lethality, which stresses the importance of this function in the mRNA decoding process.The evolutionarily conserved MnmE (TrmE) protein of Escherichia coli is a GTPase that differs extensively from regulatory GTPases such as p21 (1). Thus, MnmE exhibits a very high intrinsic GTPase hydrolysis rate and low affinity for GTP and GDP, and it can form self-assemblies. MnmE has a molecular mass of 50 kDa and is organized as a multidomain protein (see Fig. 1) consisting of an ϳ220-amino acid N-terminal domain, probably required for self-assembly, a middle GTPase domain, of about 160 residues, and an ϳ75-amino acid C-terminal domain, which contains the only Cys residue present in the protein. Strikingly, the isolated GTPase domain roughly conserves the guanine nucleotide binding and GTPase activities of the intact MnmE molecule (1).Null mnmE mutants are defective in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U 34 ) 1 (2), which is found in the wobble position (position 34) of tRNAs that read codons ending with A or G, in the mixed codon family boxes, specifically tRNAs for lysine and glutamic acid (3). The mnmA gene product (6) carries out thiolation in the 2-position of the wobble uridine (U 34 ), whereas mnmE controls the first step of the modification in the 5Ј-position, but it is unclear how many steps precede the formation of cmnm 5 s 2 U 34 (2, 4). Several data support that a second gene, named gidA, trmF, or mnmG, is also involved in the cmnm 5 group addition and that the MnmE activity precedes the activity of GidA (2, 5). The mnmC gene product has two enzymatic activities that transform the cm...

Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification

“…2). In fact, the E. coli GTPBP3 homologue (MnmE) exhibits a GTPase activity that is essential for the tRNA modifying function (27,57). For this reason, the finding that GTPBP3 may encode two isoforms differing in the length of the G domain is particularly striking.…”

“…terminal motif, which has been shown to be important for the tRNA modification function of MnmE (57). Finally, the G domain conserves the canonical Ras-like fold and includes the typical G1 to G4 motifs characteristic of all GTPases (Fig.…”

“…These properties determine that, in vitro, the GTPase cycle of MnmE proceeds efficiently without auxiliary factors (i.e., GTPase-activating proteins and guanine nucleotide exchange factors). Interestingly, the GTPase activity is essential for the tRNA modifying function of MnmE (27,57).…”

“…This is particularly interesting because exon 8A encodes 21 amino acids located between the G3 and G4 motifs of the GTPBP3 G domain. Since the GTPase activity of MnmE is essential for its tRNA modification function (27,57), the lack of exon 8A could have significant consequences for the functionality of the pro- tein encoded by variant III. Variant IV carries an additional exon 7B at the beginning of cDNA and lacks exons 1A and 8A.…”

The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy

“…To test the fidelity phenotype of the DrlmN mutant, we used a nonsense readthrough assay in which decoding of a lacZ premature stop codon by near-cognate tRNAs is required to produce b-galactosidase activity. This assay has previously proved useful to characterize the effect of several tRNA modification enzymes (Petrullo et al 1983;Elseviers et al 1984;Yim et al 2003). As shown in Table 2, mutations DmiaA (causing the loss of ms 2 i 6 A 37 in a tRNA set), and DmnmE and DmnmA (both impairing wobble uridine modifications in certain tRNAs), used here as controls, produce an error-restrictive phenotype, i.e., they lower the …”