Interplay between CedA, rpoB and double stranded DNA: A step towards understanding CedA mediated cell division in E....

Area of Science:

• Molecular Biology
• Microbiology
• Biophysics

Background:

• Cell division is essential for bacterial growth and is tightly regulated.
• DnaAcos mutant Escherichia coli exhibit compromised cell division due to chromosome over-replication.
• CedA, a DNA-binding protein, initiates cell division in these mutants through an uncharacterized mechanism.

Purpose of the Study:

• To elucidate the mechanism by which CedA, rpoB, and DNA interact to initiate cell division in E. coli.
• To understand the role of CedA-rpoB interaction in regulating cell division during chromosome over-replication.

Main Methods:

• Biophysical techniques including circular dichroism spectrometry and fluorescence spectroscopy.
• In silico analysis, including molecular docking experiments.
• BioLayer Interferometry (BLI) to determine binding affinities.

Main Results:

• Demonstrated interaction between CedA and rpoB (a subunit of RNA polymerase) using biophysical and in silico methods.
• Quantified binding affinities showing rpoB binds DNA with significantly higher affinity (K_D2 < 1.0E-12 M) than CedA (K_D2 = 9.58E-09 M).
• Docking analysis revealed more intermolecular hydrogen bonds in the rpoB-DNA complex (12) compared to the CedA-DNA complex (4).

Conclusions:

• Propose a model where chromosome over-replication signals CedA to recruit rpoB to specific DNA sites in E. coli.
• This recruitment initiates the transcription of essential cell division regulatory elements.
• The findings reveal a novel regulatory pathway for cell division initiation mediated by the CedA-rpoB-DNA complex.