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New Insights into R-loop Formation and Genome Stability in Bacteria

At the molecular level, some cellular events can have dire consequences. In bacteria, one critical issue arises during transcription when RNA can inadvertently bind to its DNA template, creating a three-stranded configuration known as an R-loop. While R-loops play essential roles in cellular functions, their misformation can lead to DNA damage, mutations, and ultimately cell death.

Recent research published in Nature Structural & Molecular Biology outlines how the enzyme RapA acts as a protective agent against the formation of R-loops in E. coli. These findings present significant implications for understanding genomic stability across different cellular systems. The research reveals that, in specific scenarios, the RNA polymerase (RNAP) responsible for transcribing DNA into RNA can promote R-loop creation, a process that is curtailed by RapA’s intervention.

“R-loops are generally detrimental, so cells have evolved various mechanisms to thwart their formation,” says Seth Darst, who leads the Laboratory of Molecular Biophysics. “Our study highlights RapA as one of these critical protective factors.”

The Mechanism of Transcription

Transcription, the process that converts DNA into RNA, is a fundamental aspect of life. In bacteria, this process initiates when RNAP binds to a DNA strand, receiving cues from sigma factors. However, the conclusion of transcription has remained less understood. Recent investigations indicate that RNAP can often stay attached to the DNA even after releasing the RNA transcript, and understanding this phenomenon has been a focus of research.

The Darst lab initially uncovered RapA in the 1990s, identifying it as an ATPase that interacts with RNAP but whose role was not clear at the time. Interest in RapA was rekindled when other researchers discovered that E. coli lacking RapA struggled to grow under high-salt stress conditions. This prompted Darst’s team to explore RapA’s functions using cryo-electron microscopy (cryo-EM) to analyze RNAP’s behavior in the presence of supercoiled DNA, which more accurately reflects the natural state of bacterial DNA compared to linear DNA.

“This study is among the first to implement negatively supercoiled DNA in cryo-EM,” states first author Joshua Brewer, who designed the experiment. “This approach helped us gain a clearer understanding of DNA topology, as well as protein interactions.”

The findings revealed that RNAP remains active following transcription termination, capable of initiating transcription again without the traditional safeguards of sigma factors. This can lead to R-loop formation unless RapA intervenes by opening the RNAP clamp, thus preventing potential genomic instability.

“Think of RNAP as a large mechanism that closes around the DNA,” Darst explains. “RapA binds to RNAP and pries open this mechanism, allowing it to detach from DNA before R-loops can form.”

Implications Beyond E. coli

The research team’s deeper dive into RapA revealed that E. coli strains engineered to lack RapA exhibited genetic instability when subjected to high-salt conditions, thus reinforcing the notion that RNAP’s propensity to form R-loops increases without RapA’s presence.

Moreover, although E. coli houses Rho, another enzyme capable of disassembling R-loops, it does not fully compensate for RapA’s absence. “Without RapA, Rho faces increased demands,” Brewer states. “This suggests that RapA and Rho function together as complementary safeguards, enhancing genome stability when bacteria encounter challenging conditions.”

The implications of this research stretch beyond E. coli, as Darst and his colleagues speculate that other organisms may possess enzymes similar to RapA that fulfill comparable roles in genomic maintenance. Identifying these mechanisms across various life forms could lead to novel strategies to address diseases linked with transcription-related genomic instability.

“We believe that other vital enzymes likely perform analogous functions throughout the tree of life,” Darst concludes. “As we uncover more about these processes, we enhance our comprehension of how cells protect their genetic integrity.”

More information: Joshua J. Brewer et al., RapA opens the RNA polymerase clamp to disrupt post-termination complexes and prevent cytotoxic R-loop formation, Nature Structural & Molecular Biology (2025). DOI: 10.1038/s41594-024-01447-8

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