Examining the behavior of RluA and TruB towards tRNA containing 2'-deoxy-2'-fluorouridine or 2'-deoxyuridine

To gain insight into substrate specificity and mechanism of pseudouridine synthases, modified stem-loop RNAs (ASL and TSL) that contain 2'-deoxy-2'-fluorouridine (Uf) or 2'-deoxyuridine (dU) were tested for their ability to be handled by the Escherichia coli &PSgr; synthases RluA and TruB.;HPLC analysis of the nucleosides from total digestion of [Uf]RNA showed no product after co-incubation with RluA or TruB. Gel shift assays provided no evidence for the formation of a covalent protein-RNA adduct when [Uf]ASL was incubated with RluA or when [Uf]TSL was incubated with TruB. HPLC analysis of [dU]ASL after incubation with RluA showed no product. After incubation of [dU]TSL with TruB, however, the analysis showed a decrease in dU, and the appearance of 2'-deoxypseudouridine, d&PSgr;, which coelutes with U, but can be detected by LC/MS.;Binding assays using 5'-33P-labeled [Uf]ASL demonstrate weak binding with RluA; Kd ≈ 11 +/- 11 muM. Similarly, binding assays using surface plasmon resonance show weak binding of [Uf]TSL with TruB; Kd ≈ 10 +/- 8.5 muM. Qualitative differences were seen in sensogram output assessing the ability of both wild-type and D64A RluA to bind ASL, [f5U]ASL and [Uf]ASL. Minimal binding was observed with wild-type or D48A TruB and [Uf]TSL. Thermodynamic analysis using isothermal titration calorimetry show differences in the titration profile of [f5U]ASL and [Uf]ASL solutions into RluA. Overall, there are slight differences in binding between the interaction of RluA with [Uf]ASL and TruB with [Uf]TSL.;RluA and TruB have similar overall folds, and the molecular surfaces are highly complementary to the shape of their respective stem-loop substrates. The weak binding between [Uf]RNA and RluA or TruB indicates a possible common and relatively non-sequence specific approach of [Uf]RNA into the active site pockets of RluA and TruB. Once this initial binding state is reached, the electrostatic repulsion between the 2'-fluoro group and the essential Asp may not permit a productive conformation for either adduct formation or catalysis. The dU in RNA bound to TruB can assume a conformation that permits isomerization, as shown by the generation of d&PSgr;. In contrast, dU in ASL cannot be positioned correctly in the RluA active site to allow isomerization.