A putative D-loop/T-loop mimic is required for structure and efficient histidylation. (A,B) SHAPE chemical probing of the wild-type TMV (A) and RMV (B) TLSs and two D-loop/T-loop mutants (boxed in figure). Reactivity was background subtracted and normalized to flanking 5′ and 3′ normalization hairpins (not shown). (C) 3H-L-histidine incorporation using S. cerevisiae HisRS of three representative TLSHis RNAs; TMV, RMV, and DiaYMV, each with the wild-type sequence as well as two D-loop/T-loop mutants. Histidylation of each RNA, as measured by covalent incorporation of 3H-L-histidine by histidine tRNA-synthetase (HisRS), is normalized to yeast tRNAHis. The dashed line and shaded region indicate the background of a reaction containing no RNA. (D) As in C, but with N. tabacum HisRS. Each reaction was performed in triplicate. Error bars represent one standard error from the mean.
