Figure 3.

The HvoTrm9–Trm112 complex catalyzes the formation of mcm⁵U on tRNAs. (A) In vitro enzymatic activity of HvoTrm9–Trm112 complex. The conditions (proteins, salt and tRNAs) used for each experiment are indicated in the table below each graph. The amount of methylated substrate (in pmol) after a 2 h reaction is indicated for every condition. Error bars have been calculated from the results of three independent experiments. (B) Reversed phase LC–MS/MS elution profiles measured from 50 pmol of synthesized mcm⁵U standard or a 2 μg injection of a total tRNA digest from WT, trm9Δ or trm112Δ strains. (C) Reversed phase LC–MS/MS elution profiles measured from 50 pmol of synthesized cm⁵U standard or a 2 μg injection of a total tRNA digest from WT, trm9Δ or trm112Δ strains. (D) SRM product ions (Supplementary Table S9) measured for the cm⁵U standard (top) and for the unknown molecule eluted 1 minute later than cm⁵U in the digested WT tRNAs sample (bottom).