The Escherichia coli trmE (mnmE) gene, involved in tRNA modification, codes for an evolutionarily conserved GTPase with unusual biochemical properties

Abstract

The evolutionarily conserved 50K protein of Escherichia coli, encoded by o454, contains a consensus GTPbinding motif. Here we show that 50K is a GTPase that differs extensively from regulatory GTPases such as p21. Thus, 50K exhibits a very high intrinsic GTPase hydrolysis rate, rather low affinity for GTP, and extremely low affinity for GDP. Moreover, it can form self-assemblies. Strikingly, the 17 kDa GTPase domain of 50K conserves the guanine nucleotide-binding and GTPase activities of the intact 50K molecule. Therefore, the structural requirements for GTP binding and GTP hydrolysis by 50K are without precedent and justify a separate classification in the GTPase superfamily. Immunoelectron microscopy reveals that 50K is a cytoplasmic protein partially associated with the inner membrane. We prove that o454 is allelic with trmE, a gene involved in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, which is found in the wobble position of some tRNAs. Our results demonstrate that 50K is essential for viability depending on the genetic background. We propose that combination of mutations affecting the decoding process, which separately do not reveal an obvious defect in growth, can give rise to lethal phenotypes, most likely due to synergism.

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

“…Genetic techniques for the construction of strains were performed as described (21). IC718 is a RZ5 derivative carrying the wild-type mnmE allele under control of promoter P tac (1). For DNA manipulations, standard procedures were followed.…”

“…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).…”

“…MnmE is also involved in modification of tRNA 1 Gln , but, in this case, the wobble base modification might be different from mnm 5 s 2 U 34 (3). In addition, the non-thiolated derivatives cmnm 5 U 34 and mnm 5 U 34 are present in tRNA 4 Leu and tRNA 4 Arg , respectively, and their formation may be also mediated by MnmE (7).…”

“…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.…”

“…Subcellular fractionation followed by immunoblotting, as well as immunoelectron microscopy, revealed that MnmE is a cytoplasmic protein partially associated with the inner membrane (1). Interestingly, the C-terminal domain of MnmE and its homologues contain the tetrapeptide motif C(I/L/V)GK at the extreme C terminus, which shows good homology with the CAAX motif (where "A" represents an aliphatic residue, and "X" represents any residue) characteristic of the Ras proteins of eukaryotic cells (14).…”