Functional complementation between mutations at two distant positions in Escherichia coli RNA polymerase as revealed...

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Subunit-subunit interactions are essential for the assembly of the core Escherichia coli RNA polymerase.
• The alpha-subunit C131A mutant is defective in contacting the beta'-subunit, despite forming an alpha2beta intermediate.

Purpose of the Study:

• To investigate the role of long-range interactions in the functional assembly of E. coli RNA polymerase.
• To identify and characterize suppressor mutations in the beta'-subunit that compensate for defects in the alpha-subunit.

Main Methods:

• Genetic screening to isolate suppressor mutations in the beta'-subunit.
• In vitro reconstitution experiments to assess subunit interactions.
• Biochemical characterization and sequence analysis of the suppressor mutation.
• Analysis of crystal structures of bacterial RNA polymerase.

Main Results:

• A suppressor mutation (G333D) was identified in the beta'-subunit, compensating for the alpha-C131A mutation.
• The suppressor mutation is allele-specific, indicating a precise functional role.
• Structural analysis revealed that the primary mutation and its suppressor are spatially distant, suggesting long-range interactions.

Conclusions:

• Long-range interactions play a significant role in the functional assembly of E. coli RNA polymerase.
• The G333D mutation in the beta'-subunit highlights the importance of conserved regions in protein assembly.