New studies highlight how the RapA enzyme helps E. coli cells defend against the harmful effects of R-loops.
From a genetic perspective, a bacterium faces a serious dilemma when newly formed RNA adheres to its DNA during transcription, creating a structure known as an R-loop. While R-loops can have beneficial roles within a cell, their formation in inappropriate locations and times can cause severe damage, resulting in DNA breaks, mutations, and ultimately cell death.
Recent findings published in Nature Structural & Molecular Biology reveal that the enzyme RapA plays a critical role in preventing R-loop formation in E. coli, with significant implications for our understanding of how cells preserve genomic integrity. The research indicates that the RNA polymerase (RNAP) enzyme, which translates DNA into RNA, can lead to excessive R-loop creation; however, this is mitigated by intervention from the protein RapA.
“R-loops are typically detrimental, so cells employ multiple backup systems to stop them from forming,” explains Seth Darst, director of the Laboratory of Molecular Biophysics. “Our discovery shows that RapA is one of those vital mechanisms we’ve been curious about for years.”
The jaws of life
All organisms depend on RNAP to convert DNA into RNA. In the case of bacteria, it’s long been established that transcription begins when RNAP attaches to a DNA strand and is activated by sigma proteins. However, the conclusion of this transcription process has remained unclear. Recent research suggests that RNAP often stays attached to the DNA even after the RNA transcript has been released — but the reasons for this, and the process behind it, were not well understood.
In the 1990s, the Darst lab identified RapA, a type of ATPase that seemed to interact with RNAP but lacked an identifiable function. “Back then, we had no idea what RapA was doing,” he recalls. However, when another research team discovered that E. coli could not thrive in stressful, high-salt environments without RapA, Darst’s interest in this enigmatic protein was rekindled.
The team utilized cryo-electron microscopy (cryo-EM) to investigate how RNAP remains bound to DNA after transcription concludes and how RapA interacts with it. They selected negatively supercoiled DNA for their experiments, which closely resembles the typically twisted form of bacterial DNA, to yield more accurate results than the linear DNA often utilized in other structural research. “This study is one of the first to employ negatively supercoiled DNA in cryo-EM experiments,” remarks first author Joshua Brewer, who designed the study. “This approach allowed us to better visualize DNA’s topological state and understand how proteins rearrange and affiliate with the DNA.”
The researchers were surprised to find that RNAP is not idle when it remains bound to DNA post-transcription. Instead, it can restart transcription without the standard protection of sigma proteins. This process can lead to the harmful formation of R-loops unless RapA acts quickly to pry open the RNAP clamp. “Think of RNAP as a large claw gripping the DNA,” Darst explains. “RapA attaches to RNAP and pries open the clamp so that it releases from the DNA, preventing the formation of R-loops.”
Beyond bacteria
As they continued their research, a clearer understanding of RapA’s function began to take shape. When bacteria lacking RapA were subjected to high-salt stress, they displayed genetic instability, suggesting that RNAP is more likely to remain bound to the DNA and generate R-loops under certain conditions.
Additionally, they discovered that although E. coli contains Rho, an enzyme known to disassemble R-loops, Rho cannot fully compensate for the absence of RapA. “Without RapA, Rho is overworked,” Brewer notes. “It seems RapA and Rho serve as complementary mechanisms — not redundant — for maintaining genomic stability when E. coli faces high-salt stress.”
The potential impact of these findings could be extensive. Darst, Brewer, and their colleagues suspect that RapA, or a similar factor that aids in releasing RNAP, is likely present not only in E. coli but also in all bacteria and potentially across all cell types. Identifying similar mechanisms in various organisms could pave the way for new strategies to tackle diseases associated with genomic instability linked to transcription issues.
“We theorize that other enzymes probably perform analogous roles throughout the evolutionary spectrum,” states Darst. “The more we uncover about these processes, the more we enhance our understanding of how cells protect their genetic material.”
