Researchers identified a new mechanism for maintaining genome stability

R-loops in the wrong place at the wrong time can be disastrous.

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In bacteria, there’s a risky situation where RNA can stick to its DNA template, forming a structure called an R-loop. While R-loops are sometimes helpful, they can also be harmful, causing DNA damage and cell death if they appear incorrectly.

Recent research by Rockefeller University has shown that an enzyme called RapA prevents R-loops formation in E. coli bacteria. This enzyme stops the RNA polymerase (RNAP) enzyme, which copies DNA into RNA, from creating too many R-loops, helping maintain the stability of the cell’s genetic information.

Generally, R-loops are bad. Hence, cells use several redundant mechanisms to prevent them from forming. Researchers discovered a protein called RapA, one of those key mechanisms.

All living things need an enzyme called RNAP to turn DNA into RNA. In bacteria, scientists have known for a long time that RNAP starts this process by attaching to a DNA strand and beginning the work when it gets a signal from proteins called sigma proteins.

However, how this process ends has been unclear. Recent studies show that RNAP often stays attached to the DNA even after it releases the new RNA, but the reasons for this were not well understood.

In the 1990s, the Darst lab found a protein called RapA, which interacts with RNAP but has no apparent purpose. “At that time, we couldn’t figure out what RapA was doing,” Darst said. However, decades later, another research team discovered that E. coli bacteria exposed to stressful, high-salt conditions could not survive without RapA. This discovery renewed Darst’s interest in the mysterious protein.

Darst’s team used a method called cryo-EM to study how the RNAP enzyme stays attached to DNA after transcribing and how the protein RapA interacts with it. They used negatively supercoiled DNA, a twisted form of DNA closer to its natural state in bacteria, rather than the usual straight DNA used in studies.

“Our study is one of the first to use this type of DNA in a cryo-EM experiment,” says Joshua Brewer, who designed the experiment. This method helped them see better how the DNA and proteins rearrange and interact with each other.

They found that RNAP doesn’t just sit idle when it stays clamped to DNA after finishing transcription. Instead, it can start transcribing again without the normal control from sigma proteins. This can create harmful R-loops without sigma unless RapA steps in and pries open the RNAP clamp.

“RNAP is like a big claw that closes around DNA,” Darst says. “RapA binds to RNAP and pulls the clamp open so it falls off the DNA before it can make R-loops.”

The role of RapA became clearer when the team put bacteria without RapA in stressful, high-salt conditions. These bacteria showed genetic instability, suggesting that RNAP is more likely to stay attached to DNA and form harmful R-loops in such situations.

They also found that while E. coli bacteria have another enzyme called Rho that can separate R-loops, Rho cannot fully take over when RapA is missing. “When RapA is gone, Rho has to work much harder,” Brewer says. RapA and Rho work together to protect the stability of the genome when E. coli faces high salt stress.

Scientists discovered that RapA plays an important role in bacteria. When bacteria that lack RapA face stressful, high-salt conditions, they experience genetic instability. This happens because RNAP tends to stick to DNA and form R-loops more often.

They also found that E. coli bacteria have another enzyme called Rho, which can help break down R-loops. However, Rho can’t fully replace RapA when RapA is missing. Instead, RapA and Rho work together to protect the genetic stability of E. coli under high salt stress.

The scientists believe that RapA, or a similar protein, might be found not just in E. coli but in all types of bacteria and possibly in all cells. Uncovering similar mechanisms in other organisms could help develop new ways to target diseases related to transcription-related genome instability.

Darst explains, “We think other enzymes probably have similar roles throughout the tree of life. The more we understand these mechanisms, the better we can learn how cells protect their genomes.”

In short, these findings could have significant implications for our understanding of genome stability and disease treatment across various organisms.

Journal Reference:

  1. Joshua J. Brewer, Koe Inlow, Rachel A Mooney, Barbara Bosch, et al. RapA opens the RNA polymerase clamp to disrupt post-termination complexes and prevent cytotoxic R-loop formation. Nature Structural & Molecular Biology. DOI: 10.1038/s41594-024-01447-8
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