[Reversion of RNA-polymerase mutations affecting F'-factor stability]

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• Amber mutations in the beta-subunit of Escherichia coli RNA polymerase lead to temperature-sensitive (TsR) phenotypes.
• These TsR mutants are viable due to partial suppression by the su2 suppressor.
• Investigating reversions helps understand gene function and regulation.

Purpose of the Study:

• To isolate and characterize Ts+ reversions of amber mutations in the beta-subunit of E. coli RNA polymerase.
• To identify the genetic locations and functional effects of these reversions.
• To elucidate the mechanisms by which these mutations restore RNA polymerase function and affect other cellular processes.

Main Methods:

• Selection of Ts+ revertants from TsR amber mutants of E. coli RNA polymerase.
• Genetic mapping of revertant mutations using bacterial genetics techniques (e.g., transduction).
• Analysis of RNA polymerase beta-polypeptide levels and phage amber mutant plating efficiency.
• Characterization of extragenic suppressor mutations and their effects on conjugation (sexduction).

Main Results:

• Three types of reversions were isolated: intragenic and two types of extragenic.
• Extragenic mutation N5 (0-15 min) restored beta-polypeptide levels and enhanced T4 phage amber mutant plating.
• Extragenic mutations D (near 64 min) acted as weak suppressors, increased su2 efficiency, and significantly decreased sexduction efficiency, suggesting an effect on episome replication.

Conclusions:

• Extragenic suppressor mutations can restore RNA polymerase function and influence phage viability.
• Mutation D exhibits pleiotropic effects, including suppression and a novel decrease in sexduction, likely via episome replication interference.
• These findings provide insights into RNA polymerase regulation and the complex interactions between bacterial genes and mobile genetic elements.