Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Bacterial topoisomerase I (TopoI) is crucial for managing DNA topology.
• Its role in regulating supercoiling during transcription is not fully understood.
• Understanding TopoI-DNA interactions is key to bacterial gene expression.

Purpose of the Study:

• To investigate the genome-wide localization and function of Escherichia coli TopoI (EcTopoI) in relation to RNA polymerase (RNAP).
• To determine the impact of the EcTopoI-RNAP interaction on DNA supercoiling and R-loop formation.
• To elucidate the mechanism by which EcTopoI prevents R-loops during transcription.

Main Methods:

• Chromatin immunoprecipitation sequencing (ChIP-Seq) to map EcTopoI and RNAP genome-wide.
• Treatment with transcription inhibitors (rifampicin) to observe enzyme relocation.
• Overexpression of EcTopoI domains and mutants to disrupt enzyme interactions.
• Pull-down assays to confirm in vivo interaction between EcTopoI and RNAP.
• Topoisomerase sequencing (Topo-Seq) to analyze DNA supercoiling.

Main Results:

• EcTopoI colocalizes with transcribing RNAP genome-wide.
• EcTopoI relocates to promoter regions upon transcription inhibition, mirroring RNAP accumulation.
• Disrupting the EcTopoI-RNAP interaction leads to increased negative supercoiling and R-loop accumulation.
• EcTopoI is enriched upstream of highly active transcription units, relieving transcription-induced negative supercoiling.
• The interaction between EcTopoI and RNAP is essential for preventing R-loops.

Conclusions:

• The interaction between EcTopoI and RNAP is vital for bacterial viability and genome integrity.
• EcTopoI actively participates in transcription regulation by relaxing negative supercoiling.
• This interaction serves as a critical mechanism to prevent the formation of harmful R-loops during active transcription.