Caracterización bioquímica y funcional de la proteína MnmE de Escherichia coli, una proteína esencial implicada en la modificación de los tRNAs.

Abstract

The Escherichia coli MnmE protein is a high evolutionarily conserved protein with GTPase activity. MnmE has a molecular mass of 50 kDa and is organized as a multidomain protein consisting of an ~220 amino acid N-terminal domain, a middle GTPase domain of about 160 residues, and a ~75 amino acid C-terminal domain, wich contains the only cysteine residue present in the protein. MnmE exhibits a very high intrinsic GTPase activity rate and low affinity for GTP and GDP and it can form self-assemblies, these unusual biochemical properties differs extensively MnmE from regulatory GTPase proteins such Ras. In vitro, the GTPase cycle works without any additional factor such GAPs or GEFs. The isolated GTPase domain roughly conserves the guanine binding and GTPase activities of intact MnmE molecule and strinkingly doesnt present any inserted activation subdomain. MnmE is a new example of a GTPase protein that follows an alternative activation mechanism without an arginine finger. MnmE is involved in the modification of the uridine at the wobble position of certain tRNAs. In this work we examine the biochemical and functional consequences of altering amino acid residues within the GTPase and C-terminal domain of MnmE. Our results indicate that the MnmE tRNA modifying function requieres effective hydrolysis of GTP and not only GTP binding as in most of the GTPases. We propose that MnmE uses the conformational change associated with GTP hydrolysis to promote the tRNAs modification reaction, in witch the C-terminal Cys may function as a catalytic residue. We also demonstrate thet point mutations abolishing the tRNA modifying function of MnmE confer synthetic lethality wich stresses the importance of the tRNA modification function in the mRNA decoding process.