(A) Sequence of TSS118 near the 3’ end of TCV gRNA. Hairpins H4a, H4b and H5 and tertiary interactions Ψ2 and Ψ3 comprise the TSS. Tertiary interactions between underlined residues are indicated by hatched arrows. Predicted contour lengths of hairpins are in brackets. Residues in green and red are moderately and highly susceptible to in-line cleavages, respectively (see C and D). Residues in grey were not evaluated. The 5A region is boxed. Green and red circles denote residues with >25% reduction or enhancement in levels of cleavage, respectively, when 5A is altered to 5U. (B) Accumulation of full-length wt and mutant TCV gRNA in Arabidopsis protoplasts. Individual adenylates were converted to the bases indicated. Data is from three independent experiments and standard deviation is shown. (C) In-line probing of the wt and 5AtoU TSS118 fragments. OH, partial hydroxide cleavage ladder; T1, partial RNase T1 digest of denatured RNA showing the location of guanylates; wt and 5AtoU control lanes were not subjected to in-line cleavage. Intensity of the in-line cleavage products correlates with flexibility of the residues. (D) Longer run of the samples shown in (C). (E) Model of TCV118 corresponding to a 25 ns state in a 40 ns-long MD trajectory. 5’ end C6 and A7 through A9 form stable Hoogsteen edge interactions with Ψ3. All 3’ end nucleotides beyond G110 remain single stranded. A fragment of this model (TSS108), comprising C5 through A112, was used as the starting point for all Steered Molecular Dynamics pulling simulations (Figure 8 and Figure 8—figure supplement 1). Nucleotides and atoms where the pulling force was applied (C5:O5’ and A112:O3’) were selected to determine the direction of pulling and have the restraints applied to are labeled and arrows indicate the direction of pulling. (F) Difference in residue flexibility in wt and 5AtoU fragments. Data is from three in-line probing experiments quantified using SAFA software.