Cytoplasmic RNA Polymerase in Escherichia coli

Area of Science:

• Bacteriology
• Molecular Biology
• Biochemistry

Background:

• Bacterial RNA polymerase (RNAP) is essential for gene transcription.
• Estimating the concentration of free, functional RNAP in the cytoplasm is crucial for understanding bacterial gene regulation.
• Previous studies have suggested that a portion of RNAP subunits may exist independently of DNA.

Purpose of the Study:

• To determine the concentration of free functional RNA polymerase in the bacterial cytoplasm.
• To investigate the localization and activity of RNA polymerase subunits in DNA-free bacterial minicells.

Main Methods:

• Quantification of RNA polymerase beta and beta' subunits in DNA-free minicells from Escherichia coli strain chi925.
• Analysis of minicell infectivity with bacteriophage T7.
• In vitro transcription assays using RNA polymerase subunits extracted from minicell lysates.

Main Results:

• RNA polymerase constituted 1.0% of total protein in E. coli grown under optimal conditions.
• RNA polymerase beta and beta' subunits in minicells represented approximately 17% (2.5 microM) of the concentration found in whole cells.
• Infection with bacteriophage T7 resulted in abortive infection in 80% of minicells.
• Extracted RNA polymerase subunits from minicells showed no detectable activity in vitro and were associated with minicell envelopes.

Conclusions:

• Most functional bacterial RNA polymerase is associated with DNA.
• Little to no free functional RNA polymerase segregates into DNA-free minicells.
• The association of RNA polymerase with DNA is critical for its transcriptional activity.