The Escherichia coli trmE (mnmE) gene, involved in tRNA modification, codes for an evolutionarily conserved GTPase with unusual biochemical properties.
The evolutionarily conserved 50K protein of Escherichia coli, encoded by o454, contains a consensus GTP-binding 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.
Cabedo
H
H
Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Valencia 46010, Spain.
Macián
F
F
Villarroya
M
M
Escudero
J C
JC
Martínez-Vicente
M
M
Knecht
E
E
Armengod
M E
ME
D001483:Base Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial / Q000235:genetics*; D016384:Consensus Sequence; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000648:ultrastructure; D019143:Evolution, Molecular; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics*
D046911:Macromolecular Substances; D012329:RNA, Bacterial; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D020558:GTP Phosphohydrolases