Crystal structures of the tRNA:m 2 G6 methyltransferase Trm14/TrmN from two domains of life

Abstract

Methyltransferases (MTases) form a major class of tRNA-modifying enzymes needed for the proper functioning of tRNA. Recently, RNA MTases from the TrmN/Trm14 family that are present in Archaea, Bacteria and Eukaryota have been shown to specifically modify tRNA ^Phe at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m ² G6 ( N² -methylguanosine) MTase _TTC TrmN from Thermus thermophilus and its ortholog _Pf Trm14 from Pyrococcus furiosus . Structures of _Pf Trm14 were solved in complex with the methyl donor S -adenosyl- l -methionine (SAM or AdoMet), as well as the reaction product S -adenosyl-homocysteine (SAH or AdoHcy) and the inhibitor sinefungin. _TTC TrmN and _Pf Trm14 consist of an N-terminal THUMP domain fused to a catalytic Rossmann-fold MTase (RFM) domain. These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis. Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain. This is further supported by a docking model of TrmN in complex with tRNA ^Phe of T. thermophilus and via site-directed mutagenesis.