Mg2+-induced proton release from Escherichia coli ribosome and ribosomal RNA

Escherichia coli ribosome released protons upon addition of Mg2+. The Mg2+-induced proton release was studied by means of the pH-stat technique. The number of protons released from a 70 S ribosome in the Mg2+ concentration range 1-20 mM was about 30 at pH 7 and 7.6, and increased to about 40 at pH 6.5. The rRNA mixture extracted from 70 S ribosome showed proton release of amount and of pH dependence similar to those of the 70 S ribosome but the ribosomal protein mixture released few. This indicates that rRNA is the main source of the protons released from ribosome. The pH titration of rRNA showed that the pKa values of nucleotide bases were downward shifted upon Mg2+ binding. This pKa shift can account for the proton release. The Scatchard plots of proton release from rRNA and ribosome were concave upward, showing that the Mg2+-binding sites leading to proton release were either heterogeneous or had a negative cooperativity. A model assuming heterogeneous Mg2+-binding sites is shown to be unable to explain the proton release. Electrostatic field effect models are proposed in which Mg2+ modulates the electrostatic field of phosphate groups and the potential change induces a shift of the pKa values of bases that leads to the proton release. These models can explain the main features of the proton release.