Figure 2.

Promiscuous ligand binding by the B. subtilis yvrC cobalamin riboswitch. Co-transcriptional ligand loading was an effective approach in generating relatively homogenous samples of cobalamin-bound riboswitches, as demonstrated by both electrophoretic mobility shifts and size-exclusion chromatography (SEC) with multi-angle light scattering (MALS). (A) Left: The E. coli cobalamin riboswitch is specific for adenosylcobalamin (A, red), whereas the B. subtilis cobalamin riboswitch binds to adenosylcobalamin, hydroxocobalamin (H, green), and methylcobalamin (M, purple). Subtle gel migration differences were observed between the adenosylcobalamin versus hydroxocobalamin/methylcobalamin-bound B. subtilis riboswitch samples (green line). As expected, neither riboswitch associates with cyanocobalamin, a synthetic cobalamin derivative (C, orange, and C*, yellow, correspond to two different preparations). The electrophoretic mobility shift patterns of the RNAs were the same when there is no ligand (ᴓ, black) and when co-transcribed with cyanocobalamin. Right: Denaturing gel in 8 M urea shows that differences in native gel migration were not due to RNA sample degradation. (B) The B. subtilis cobalamin riboswitch exhibits conformational variation when bound to different cobalamin derivatives. SEC-MALS analysis shows that the B. subtilis cobalamin riboswitch binds to adenosylcobalamin, hydroxocobalamin, and methylcobalamin primarily as an RNA dimer. The differences observed in the various elution peak positions (vertical arrows) probably reflect conformational variation in the adenosylcobalamin versus the hydroxocobalamin/methylcobalamin-bound forms of the riboswitch. No difference in elution time was observed between the B. subtilis cobalamin riboswitch without ligand and with cyanocobalamin, consistent with the native gel findings.