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Mg2+-induced proton release from Escherichia coli ribosome and ribosomal RNA
Abstract:
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.
Insights
Magnesium ions (Mg2+) trigger proton release from Escherichia coli ribosomes, primarily due to ribosomal RNA (rRNA). This Mg2+-induced proton release is explained by electrostatic models affecting nucleotide base pKa values.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Ribosomes are essential molecular machines responsible for protein synthesis.
- Magnesium ions (Mg2+) play crucial roles in ribosome structure and function.
- Proton release from ribosomes upon Mg2+ addition has been observed but not fully explained.
Purpose of the Study:
- To investigate the source and mechanism of Mg2+-induced proton release from Escherichia coli ribosomes.
- To elucidate the role of ribosomal RNA (rRNA) and ribosomal proteins in this phenomenon.
- To develop models explaining the observed proton release based on Mg2+ binding.
Main Methods:
- Utilized the pH-stat technique to quantify proton release.
- Studied proton release from intact 70S ribosomes, extracted rRNA, and ribosomal proteins.
- Performed pH titration of rRNA in the presence of Mg2+.
- Analyzed Mg2+ binding using Scatchard plots.
Main Results:
- Escherichia coli ribosomes released protons upon Mg2+ addition, with the amount varying with pH.
- Ribosomal RNA (rRNA) was identified as the primary source of released protons, not ribosomal proteins.
- Mg2+ binding to rRNA caused a downward shift in nucleotide base pKa values, accounting for proton release.
- Scatchard plots indicated heterogeneous or negatively cooperative Mg2+ binding sites, ruling out simple heterogeneous models.
Conclusions:
- Mg2+-induced proton release from E. coli ribosomes is mainly mediated by rRNA.
- Electrostatic field effect models, where Mg2+ modulates phosphate group interactions and shifts base pKa values, successfully explain the observed proton release.
- This work provides insights into the fundamental interactions governing ribosome function and Mg2+ binding.