How far do H/ACA sRNPs contribute to rRNA pseudouridylation in Archaea was still an open question. Hence here, by computational search in three Pyrococcus genomes, we identified seven H/ACA sRNAs and predicted their target sites in rRNAs. In parallel, we experimentally identified 17 Ψ residues in P. abyssi rRNAs. By in vitro reconstitution of H/ACA sRNPs, we assigned 15 out of the 17 Ψ residues to the 7 identified H/ACA sRNAs: one H/ACA motif can guide up to three distinct pseudouridylations. Interestingly, by using a 23S rRNA fragment as the substrate, one of the two remaining Ψ residues could be formed in vitro by the aCBF5/aNOP10/aGAR1 complex without guide sRNA. Our results shed light on structural constraints in archaeal H/ACA sRNPs: the length of helix H2 is of 5 or 6 bps, the distance between the ANA motif and the targeted U residue is of 14 or 15 nts, and the stability of the interaction formed by the substrate rRNA and the 3′-guide sequence is more important than that formed with the 5′-guide sequence. Surprisingly, we showed that a sRNA–rRNA interaction with the targeted uridine in a single-stranded 5′-UNN-3′ trinucleotide instead of the canonical 5′-UN-3′ dinucleotide is functional.

The 15.5-kD protein and its yeast homolog Snu13p bind U4 snRNA, U3 snoRNA, and the C/D box snoRNAs. In U4 snRNA, they associate with a helix-bulge-helix (K-turn) structure. U3 snoRNA contains two conserved pairs of boxes, C/D and B/C, which were both expected to bind the 15.

A density functional study of the hydrolysis reaction of phosphodiesters with a series of attacking nucleophiles in the gas phase and in solution is presented. The nucleophiles HOH, HO-, CH3OH, and CH3O- were studied in reactions with ethylene phosphate, 2'3'-ribose cyclic phosphate and in their neutral (protonated) and monoanionic forms. Stationary-point geometries for the reactions were determined at the density functional B3LYP/6-31++G(d,p) level followed by energy refinement at the B3LYP/6-311++G(3df,2p) level. Solvation effects were estimated by using a dielectric approximation with the polarizable continuum model (PCM) at the gas-phase optimized geometries. This series of reactions characterizes factors that influence the intrinsic reactivity of the model phosphate compounds, including the effect of nucleophile, protonation state, cyclic structure, and solvent. The present study of the in-line mechanism for phosphodiester hydrolysis, a reaction of considerable biological importance, has implications for enzymatic mechanisms. The analysis generally supports the associative mechanism for phosphate ester hydrolysis. The results highlight the importance for the reaction barrier of charge neutralization resulting from the protonation of the nonbridging phosphoryl oxygens and the role of internal hydrogen transfer in the gas-phase mechanism. It also shows that solvent stabilization has a profound influence on the relative barrier heights for the dianionic, monoanionic, and neutral reactions. The calculations provide a comprehensive data set for the in-line hydrolysis mechanisms that can be used for the development of improved semiempirical quantum models for phosphate hydrolysis reactions.