Selective terminal methylation of a tRNA wobble base. Issue 7 (18th January 2018) - Masuda, Isao
Abstract: Active tRNAs are extensively post-transcriptionally modified, particularly at the wobble position 34 and the position 37 on the 3′-side of the anticodon. The 5-carboxy-methoxy modification of U34 (cmo 5 U34) is present in Gram-negative tRNAs for six amino acids (Ala, Ser, Pro, Thr, Leu and Val), four of which (Ala, Ser, Pro and Thr) have a terminal methyl group to form 5-methoxy-carbonyl-methoxy-uridine (mcmo 5 U34) for higher reading-frame accuracy. The molecular basis for the selective terminal methylation is not understood. Many cmo 5 U34-tRNAs are essential for growth and cannot be substituted for mutational analysis. We show here that, with a novel genetic approach, we have created and isolated mutants of Escherichia coli tRNA Pro and tRNA Val for analysis of the selective terminal methylation. We show that substitution of G35 in the anticodon of tRNA Pro inactivates the terminal methylation, whereas introduction of G35 to tRNA Val confers it, indicating that G35 is a major determinant for the selectivity. We also show that, in tRNA Pro, the terminal methylation at U34 is dependent on the primary m 1 G methylation at position 37 but not vice versa, indicating a hierarchical ranking of modifications between positions 34 and 37. We suggest that this hierarchy provides a mechanism to ensure top performance of a tRNA inside of cells.