Characterization of two homologous 2′-O-methyltransferases showing different specificities for their tRNA substrates

Abstract

The 2 ′ -O-methylation of the nucleoside at position 32 of tRNA is found in organisms belonging to the three domains of life. Unrelated enzymes catalyzing this modification in Bacteria (TrmJ) and Eukarya (Trm7) have already been identified, but until now, no information is available for the archaeal enzyme. In this work we have identified the methyltransferase of the archaeon Sulfolobus acidocaldarius responsible for the 2 ′ -O-methylation at position 32. This enzyme is a homolog of the bacterial TrmJ. Remarkably, both enzymes have different specificities for the nature of the nucleoside at position 32. While the four canonical nucleosides are substrates of the Escherichia coli enzyme, the archaeal TrmJ can only methylate the ribose of a cytidine. Moreover, the two enzymes recognize their tRNA substrates in a different way. We have solved the crystal structure of the catalytic domain of both enzymes to gain better understanding of these differences at a molecular level.

Identification of determinants for tRNA substrate recognition byEscherichia coliC/U34 2′-O-methyltransferase

“…38 EcTrmJ needs the full-length tRNA and correct identity elements within the D stem and loop region, which is totally different from TrmL. 39 Our data showed that 2 0 -O-methylation at the wobble position by TrmL requires a previous i 6 A37 modification. It is, however, unclear how the i 6 A37 modification influences tRNA recognition by TrmL.…”

“…38,39 E. coli TrmJ (EcTrmJ) requires full-length tRNA molecules as substrates. 39 The elements of tRNA required for recognition and methylation by TrmL have not yet been identified. The mechanism by which TrmL localizes to the anticodon domain of the large L-shaped structure and distinguishes C/U in the wobble position from the other nucleotides is an intriguing question.…”

Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response inPseudomonas aeruginosa

“…This motif has previously been shown to be critical for binding of SAM/SAH and tRNA (33,34). SAM becomes SAH (S−Adenosyl Homocysteine) after losing its methyl group in the methylation reaction.…”

Two-subunit enzymes involved in eukaryotic post-transcriptional tRNA modification

“…[94][95][96][97] The formation of Nm 32 in E. coli and in the archaeon Sulfolobus acidocaldarius requires members of the homodimeric TrmJ SPOUT methyltransferase family, 88,89 which are not obviously related to Trm7, Trm732, or Trm734. Ec TrmJ appears to require elements in the D-stem and loop for modification activity, whereas Sa TrmJ appears to require elements solely in the anticodon loop.…”

“…Ec TrmJ appears to require elements in the D-stem and loop for modification activity, whereas Sa TrmJ appears to require elements solely in the anticodon loop. 89 The formation of Nm 34 requires distinct genes in bacteria and archaea, neither of which are related to components of the TRM7 modification machinery. Thus, in E. coli, Cm 34 and Um 34 on certain tRNA Leu species are formed by the SPOUT methyltransferase TrmL, which recognizes its substrates by interactions with specific residues, including the N 6 -(isopentenyl)-2-methylthioadenosine modification formed from A 37 in the anticodon loop of substrate tRNAs.…”