[Influence of ionic strength on RNA-polymerase structure]

Area of Science:

• Molecular Biology
• Biochemistry
• Enzymology

Background:

• RNA polymerase holoenzyme is crucial for transcription initiation.
• The holoenzyme consists of core enzyme and sigma-subunit.
• Enzyme structure and function are influenced by environmental conditions like ionic strength.

Purpose of the Study:

• To investigate the effect of ionic strength on RNA polymerase holoenzyme stability and subunit association.
• To explore the relationship between enzyme dimerization and sigma-subunit dissociation.
• To determine the optimal ionic strength for holoenzyme reconstitution.

Main Methods:

• DNA-agarose chromatography was used to analyze RNA polymerase holoenzyme composition.
• Ionic strength was varied using potassium chloride (KCl) concentrations.
• Reconstitution experiments were performed using purified core enzyme and sigma-subunit.

Main Results:

• Holoenzyme stability decreased with decreasing ionic strength (0.15 to 0.05 M KCl), with a 50% loss of holoenzyme.
• Free sigma-subunit appeared at lower ionic strengths, coinciding with enzyme dimer-monomer transitions.
• Enzyme dimerization appears to follow the equilibrium: E sigma + E sigma <=> E2 sigma.
• Reconstitution of holoenzyme from core enzyme and sigma-subunit was inefficient below 0.1 M KCl and successful at 0.2 M KCl and above.

Conclusions:

• Ionic strength significantly impacts RNA polymerase holoenzyme integrity, promoting sigma-subunit dissociation and enzyme monomerization at low salt concentrations.
• Enzyme dimerization may play a role in promoter searching and the regulation of RNA synthesis.
• Specific ionic conditions are necessary for the successful reconstitution of functional RNA polymerase holoenzyme.