FIGURE 2.

Divergent TLS^Val RNAs containing stem–loop insertions are competent substrates for valylation. (A,B) Chemical probing of TLS representatives from Peanut clump virus (A) and Nudaurelia capensis beta virus (B) using the SHAPE reagent NMIA. Reactivity was background subtracted and normalized according to the reactivity of loop regions in hairpin structures (not shown) flanking the TLS structure on both the 5′ and 3′ ends. (C) Activity of valyl tRNA synthetase (ValRS) on TLS RNAs as measured by the covalent addition of radiolabeled (³H) valine at their 3′ termini. The ³H incorporation, as measured by a scintillation counter, was normalized to the TYMV TLS^Val construct, which had been previously tested and optimized under these reaction conditions. The truncated NCBV construct begins at nucleotide C15 (see panel B for sequence; the dashed box indicates the deleted region). The BMV TLS belongs to a separate class of tyrosine-accepting TLSs. Orange and blue bars correspond to the color schemes for PCV and NCBV, respectively, in panels A and B as well as in Figure 4.