Structure-function relationships in Escherichia coli transcription termination protein Rho revealed by radiation...

Area of Science:

• Molecular Biology
• Biophysics
• Enzymology

Background:

• Escherichia coli transcription termination factor Rho is a homohexameric enzyme.
• Understanding Rho's functional domains and subunit interactions is crucial for elucidating transcription termination mechanisms.

Purpose of the Study:

• To determine the target sizes for inactivation of Rho's RNA-dependent ATPase activity, ATP binding, and physical destruction using high-energy electrons.
• To investigate the role of subunit interactions in Rho's ATPase activity and stability.

Main Methods:

• Irradiation of Rho with high-energy electrons.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis to assess polypeptide destruction.
• Analysis of inactivation kinetics to determine target sizes for different functions.

Main Results:

• Physical destruction and ATP binding inactivation occurred with a target size of a dimer, suggesting energy transfer between subunits.
• RNA-dependent ATPase activity inactivation indicated a target size of a trimer to tetramer, implying subunit interactions are essential for hydrolysis.
• Modeling suggested a hexamer target size with dimers as exchanging units for ATPase activity, consistent with prior studies.

Conclusions:

• Rho's functional inactivation is dependent on the specific activity, with different target sizes observed for physical destruction, ATP binding, and RNA-dependent ATPase activity.
• Subunit interactions, potentially involving dimers, are critical for Rho's RNA-dependent ATPase activity.
• The findings provide insights into the molecular mechanisms underlying transcription termination by Rho.