Fundamental processes that occur along strands of DNA, including RNA transcription and DNA replication, commonly encounter obstacles – or “roadblocks” – that can impede progress and ultimately result in mutations and/or DNA damage.
The bacterial protein Mfd (mutation frequency decline) is best known for its ability to free a stalled RNA polymerase (RNAP) in transcription-coupled repair. And there is mounting evidence that Mfd is good at helping RNAP to resolve other roadblocks beyond this repair function. However, the mechanism by which it does these isn’t well understood.
Using techniques it developed, a group led by Michelle Wang, Cornell physics professor and Howard Hughes Medical Institute investigator, has uncovered a key property of this important protein that explains its ability to selectively remove certain roadblocks to keep fundamental processes moving.
The group published a paper, “Mfd Dynamically Regulates Transcription via a Release and Catch-up Mechanism,” Dec. 7 in the journal Cell. The lead author is postdoctoral researcher Tung Le of the Wang Lab.
DNA transcription is initiated when the RNAP binds to the DNA. This most fundamental of chromosomal processes decodes the genetic code – the “blueprint” – for cells, but there are numerous functions occurring simultaneously on the DNA molecule, some more important than others.
“RNAPs can be stalled by roadblocks such as other DNA-bound proteins,” Wang said.
Mfd is widely recognized for its ability to free a stalled RNAP, but why it’s able to selectively target stalled RNAPs is a mystery, especially because of the math: In each E. coli cell, there are a few thousand copies of RNAP but only a few hundred copies of Mfd.
And because a stalled RNAP doesn’t look much different from an elongating one, the mechanism by which Mfd can differentiate the two and act accordingly has been unclear.
Wang and her group made a couple of important findings related to Mfd, enabled by innovative single molecule tracking methods. The first: Mfd moves on its own along the DNA strand.
“We found that it moves very steadily and at a pretty good speed,” Wang said. “That took us by surprise.”
So instead of having to locate a stalled RNAP in a three-dimensional space, it can just move along the DNA until it runs into one. That led to the second finding: RNAP moves faster along the DNA than Mfd – think of two cars at a stoplight, and the car in front moves away quicker than the one behind. But when the RNAP hits a “roadblock,” the Mfd catches up and acts on it.
The Mfd will either release the RNAP from the roadblock or, if the obstacle is too great, dissociate the RNAP, which clears the DNA for other important processes. And due to its own limited processivity, Mfd ultimately breaks away from DNA and avoids becoming a roadblock itself.
These findings illustrate a remarkably delicate coordination between Mfd and RNAP, which allows for efficient targeting and recycling of Mfd and expedient conflict resolution.
Wang said two new tracking techniques developed during this research, used in combination with other techniques the lab previously developed, could be used to track the movement of other motor proteins along DNA.
Other contributors included members of the Wang lab and labs of collaborators, along with Professor Jeffrey Roberts of the Department of Molecular Biology and Genetics at Cornell and Professor Alexandra Deaconescu of Brown University.
Primary support for this research came from the Howard Hughes Medical Institute, the National Science Foundation, and the National Institutes of Health.