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Biochemistry. 2017 May 23;56(20):2537-2548. doi: 10.1021/acs.biochem.7b00174. Epub 2017 May 9.

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

Author information

1
Department of Chemistry, Pennsylvania State University , University Park, Pennsylvania 16802, United States.
2
Center for RNA Molecular Biology, Pennsylvania State University , University Park, Pennsylvania 16802, United States.
3
Department of Biochemistry and Molecular Biology, Pennsylvania State University , University Park, Pennsylvania 16802, United States.

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 pKa 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 Mg2+ ions. We introduce global fitting for analyzing ribozyme rate-pH data parametric in Mg2+ 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 pKa 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 Mg2+ ion. The value for the pKa of the -1 nucleotide, C17, is increased a remarkable 3.5-5 pKa units toward neutrality. Shifting of the pKa 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 Mg2+ 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.

PMID:
28485924
DOI:
10.1021/acs.biochem.7b00174
[Indexed for MEDLINE]

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