The GTPase activity and C-terminal cysteine of the Escherichia coli MnmE protein are essential for its tRNA modifying function.
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.
Laboratorio de Genética Molecular and Laboratorio de Biología Celular, Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Amadeo de Saboya 4, Valencia 46010, Spain.
Lucía
L
Yim
Marta
M
Martínez-Vicente
Magdalena
M
Villarroya
Carmen
C
Aguado
Erwin
E
Knecht
María-Eugenia
ME
Armengod
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D003545:Cysteine; D004926:Escherichia coli / Q000737:chemistry*; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation / Q000187:drug effects; D012343:RNA, Transfer / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D009711:Nucleotides; D010446:Peptide Fragments; C417279:TRME protein, Thermotoga maritima; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D003545:Cysteine