Transfer RNA Recognition Mechanism of Thermoplasma acidophilum Trm56, a SPOUT tRNA Methyltransferase that Possesses an Unusually Long C-terminal Region

Abstract

Trm56 from Thermoplasma acidophilum is an exceptional SpoU-TrmD superfamily enzyme, which possesses an unusually long C-terminal region, and catalyzes methylation of 2′-OH of ribose of C56 in tRNA. T. acidophilum Trm56 methylates elongator tRNA^Met (tRNA^Mete) transcript effectively, however this enzyme methylates tRNA^Leu transcript slowly. This enzymatic feature is common in T. acidophilum Trm56 and Pyrococcus horikoshii Trm56, which possesses a short C-terminal region. Kinetic analysis revealed that Km value of T. acidophilum Trm56 for tRNA^Leu is larger than that for tRNA^Mete. Our analysis of native tRNAs revealed that the 2′-O-methylcytidine content at position 56 (Cm56) in tRNA^Leu is 1.9% while in tRNA^Mete it is 63.1%, showing that the in vitro enzymatic properties of T. acidophilum Trm56 are reflected in the Cm56 modification levels in living cells. Experiments with chimera tRNA transcript of tRNA^Leu and tRNA^Mete showed that T. acidophilum Trm56 recognizes the T-loop and the three-dimensional core of tRNA. Furthermore, experiments with mutant tRNA^Mete transcript revealed that the G53-C61 base pair and U54U55C56purine57purine58 sequence are essential for methylation. Crystal structures of apo- and 5′-methyl-5′-thioadenosine binding forms of the catalytic domain of T. acidophilum Trm56 revealed that the structure of the 5′-methyl-5′-thioadenosine binding pocket and overall structure of the catalytic domain of T. acidophilum Trm56 closely resemble those of P. horikoshii Trm56. Experiments with a chimera of T. acidophilum and P. horikoshii Trm56 proteins demonstrates that the catalytic domain of T. acidophilum Trm56 is responsible for the slow methylation of tRNA^Leu transcript.