Cooperative Interactions in the Hammerhead Ribozyme Drive pK_a Shifting of G12 and Its Stacked Base C17

Abstract

General acid-base catalysis is a key mechanistic strategy in protein and RNA enzymes. Ribozymes use hydrated metal ions, nucleobases, and organic cofactors to carry this out. In most small ribozymes, a guanosine is positioned to participate in proton transfer with the nucleophilic 2'-OH. The unshifted pK values for nucleobases and solvated metal ions are far from neutrality, however, and thus nonideal for general acid-base catalysis. Herein, evidence is provided for cooperative interaction in the hammerhead ribozyme among the guanine that interacts with the nucleophilic 2'-OH, G12, the -1 nucleobase C17, and Mg ions. We introduce global fitting for analyzing ribozyme rate-pH data parametric in Mg concentration and benchmark this method on data from the hepatitis delta virus ribozyme. We then apply global fitting to new rate-pH data for the hammerhead ribozyme using a minimal three-dimensional, four-channel cooperative model. The value for the pK of G12 that we obtain is channel-dependent and varies from 8.1 to 9.9, shifting closest toward neutrality in the presence of two cationic species: C17H and a Mg ion. The value for the pK of the -1 nucleotide, C17, is increased a remarkable 3.5-5 pK units toward neutrality. Shifting of the pK of C17 appears to be driven by an electrostatic sandwich of C17 between carbonyl groups of the 5'-neighboring U and of G12 and involves cation-π interactions. Rate-pH profiles reveal that the major reactive channel under biological Mg and pH involves a cationic C17 rather than a second metal ion. Substitution of a cationic base for a metal underscores the versatility of RNA.

Incorporating uracil and 5-halouracils into short peptide nucleic acids for enhanced recognition of A–U pairs in dsRNAs

“…Our results also suggest that one may incorporate a single Br U or multiple nonconsecutive Br U residues into C base-containing PNAs to enhance PNA·dsRNA triplex formation and reduce pH dependence. The reduced pH dependence may be due to an upshift of the apparent pK a of C bases through favorable stacking interactions (78,79) with halouracil bases. It is significant that incorporating a Br UL step in a PNA (from N-terminus to C-terminus) can coop-Nucleic Acids Research, 2018, Vol.…”

“…Compared to P1, the singly-and doubly-modified PNA sequences have higher melting temperatures (based on the heating curves), with Cl U and Br U modifications being the most stabilizing ( Figure 4C, D). Compared to F U base, Cl U and Br U may be more ideal for optimizing the coupled stacking and hydrogen bonding interactions (78,79) for the sequences tested here, with the PNAs containing pyrimidine residues only. The stabilization effect of X U substitution on PNA-RNA duplex was also observed previously for a different PNA sequence (with the X U residue flanked by two G residues), although relatively less variation among F U, Br U, and Cl U was observed 5 (F, H).…”

Nucleic Acid Catalysis under Potential Prebiotic Conditions

“…However, the free form of the four canonical nucleobases have p K a values far from neutrality and are therefore suboptimal for general acid‐base catalysis . In some ribozymes, the local molecular environment can cause a considerable shift in the p K a of both general acid and base towards neutrality, a similar effect to that found in some proteins . If both ionizable groups are sufficiently perturbed, the pH dependence of catalytic rates shows a “bell‐shaped” pH rate profile, where the rates are maximal around pH 7 .…”

“…[125] In somer ibozymes, the local molecular environment can cause ac onsiderable shift in the pK a of both general acid and base towards neutrality,asimilare ffect to that found in some proteins. [126][127][128] If both ionizable groupsa re sufficientlyp erturbed, the pH dependence of catalytic rates showsa"bell-shaped" pH rate profile, where the rates are maximal aroundp H7. [123,129] In other cases,s uch as for the hairpin (HP) ribozyme, the rates of RNA cleavage (and ligation) increaseu pt op H7,b ut plateau at higherv alues due to the high pK a of N1 in the catalytically activeguanosine base.…”

Structural Simplicity and Mechanistic Complexity in the Hammerhead Ribozyme

“…Two recent publications argue that even the comparatively wellunderstood mechanism for how the general base facilitates catalysis in the cleavage reaction of the hammerhead ribozyme may in fact be far more complex than previously appreciated. 30,31 It is proposed that the cleavagesite nucleotide, C17, becomes protonated, and therefore positively charged, at physiological pH, and that in the transition-state, cation-π interactions between G12 À (the deprotonated general base), C17H + (the protonated cleavage-site nucleotide), and U16.1 (the invariant nucleotide 5 0 to C17), cooperatively stabilize the catalytically relevant form of the general base.…”