Contribution of two conserved histidines to the dual activity of archaeal RNA guide-dependent and -independent pseudouridine synthase Cbf5

Two histidine residues in Cbf5 are proposed to contribute in the RNA-guided and unguided system. (A) Schematic views of the guide RNA-independent activity of Cbf5. (A₁) A 2D representation of a substrate tRNA is shown with the target U₅₅ in the TΨC arm. (A₂) Details of the tRNA TΨC arm showing recognition of the U₅₄:A₅₈ reverse Hoogsteen base pair by histidine H43 (the counterpart of H77 in Cbf5), as identified in the cocrystal structure of the E. coli TruB in complex with the tRNA (Hoang and Ferré-D'Amaré 2001). (B) Schematic views of the guide RNA-dependent activity of Cbf5. (B₁) Scheme of an archaeal box H/ACA sRNP showing the different proteins which are represented by circles, associated with the guide RNA folded as single hairpin (in black). The substrate RNA in red is shown associated with the RNP enzyme by forming two base-pair interactions with the guide RNA (PS1 and PS2). The ACA box and the K-turn/K-loop motif are indicated. (B₂) Schematic representation of the PS2 helix formed by base-pairing of the substrate RNA with the 3′ strand of the pseudouridylation pocket of the guide RNA and ending with a N:N′-1 base pair (N from the guide RNA, N′-1 from the substrate RNA). Histidine H60 but not H77 was found to stack over nucleobase A or C, corresponding to nucleotide N′-1, in the crystal structures of two distinct H/ACA sRNPs bound to a substrate RNA (Duan et al. 2009; Liang et al. 2009). (C) Structural alignments showing respective position of the conserved histidines in substrate-bound H/ACA sRNP enzymes. The numbering of histidines is from Pyrococcus furiosus (H63 and H80 correspond to, respectively, H60 and H77 in P. abyssi). The 3D structures were superposed with Swiss protein viewer and representation prepared with PyMol. (C₁) Superposition of two H/ACA sRNPs by using the PDB access number 3HAX (Duan et al. 2009) and 3HJW (Liang et al. 2009). The histidines are in purple and the nucleobases of the substrate are in brown and of the RNA guide in sand. (C₂) Superposition of TruB (PDB 1K8W [Hoang and Ferré-D'Amaré 2001]) with the H/ACA sRNP enzymes. Histidine H43 is in orange and the U₅₄:A₅₈ reverse Hoogsteen in salmon.