Fig. 3.

Selective recognition of 2′-dG by a U,C,C group of guanine riboswitch variants. (A) Consensus sequence and secondary structure of a putative purine riboswitch variant identified via the bioinformatics strategy described in this report. Boxed annotations indicate differences from guanine riboswitches. Other annotations are as described for Fig. 2A. (B) Sequence and secondary structure of the 71 env-23 RNA. Regions of constant, increasing, and decreasing internucleotide cleavage were determined from the in-line probing data presented in C. The arrowhead indicates the start of this data. Lowercase g letters identify guanosine nucleotides encoded by the template to facilitate efficient RNA production by in vitro transcription. Numbers 1 through 5 identify regions that undergo 2′-dG–dependent structure modulation. (C) Denaturing (8 M urea) PAGE analysis of in-line probing reactions of 5′-³²P-labeled 71 env-23 RNA in the presence of 100 µM deoxyguanosine (2′-dG), 100 µM guanine (g), or in the absence of ligand (‒). NR, T1, and ⁻OH indicate no reaction, partial digestion with RNase T1 (cleaves after G residues), and partial digestion with hydroxide (cleaves after every residue). Several RNase T1 product bands are labeled. Regions undergoing structural modulation (1 through 5) and predicted stems (P1 through P3) are indicated. (D) Plot of the fraction of RNA bound to ligand vs. the logarithm of the molar concentration (c) of 2′-dG. Data are derived from SI Appendix, Fig. S1. Included are a theoretical binding curve expected for a one-to-one interaction between ligand and RNA for the indicated K_D value. (E) Plot of the dissociation constants measured for various analogs of 2′-dG for the 71 env-23 RNA (Left). List of compounds that resulted in no structural modulation of the 71 env-23 RNA upon addition at the indicated concentrations (Right). 3-(2-Deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one is abbreviated M1G.