Discovery of Subversive Textbooks: Cryo-EM Reveals the Story behind Transcriptio

Posted by Jerry Carter on December 25th, 2020

During the transcription stage, RNA needs to terminate at the right place. Who is responsible for this process? Fifty years ago, scientists speculated about a possible model: a hexameric ring-shaped RecA family RNA translocator ρ (Rho), which can first bind to RNA and participate in the transfer of ATP-driven RNA polymerase to RNA. When RNA polymerase stops, ρ helps specific RNA fragments to leave the DNA. This is also the explanation given to us in the textbook. However, some researchers have realized that there is insufficient evidence for this claim, and the mechanism of transcription termination is still unknown.

Recently, the team of Markus C. Wahl of the Free University of Berlin and the team of Irina Artsimovitch of Ohio State University jointly published a research paper entitled Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ in Science. In this research paper, the author uses cryo-electron microscopy (cyro-EM) single particle analysis technology to analyze the structure of the transcription complex formed by ATPase ρ during its functioning, and captures the image of RNA polymerase translocation on E. coli DNA , revealing the true process of ρ terminating gene expression, once again deepening our precise understanding of the transcription process.

Because ρ can disintegrate the complex in minutes or even seconds, this complex is very difficult to obtain. The researchers cleverly used cryo-electron microscopy to capture images of RNA polymerase running on the DNA template in E. coli before the complex disintegrated. The results indicate that RNA polymerase (RNAP), transcription factors NusA and NusG play a key role in the path of ρ-mediated elongation complex (EC) disintegration.

The author reveals how ρ acts on a complete transcription complex composed of RNA polymerase and two accessory proteins that accompany the transcription process, from the beginning of the binding of ρ with RNA polymerase to the end of RNA polymerase losing control of nucleic acids.

First, an open ρ loop contacts with NusA, NusG, and multiple regions of RNA polymerase to capture and partially untie the proximal and upstream DNA. Then, NusA wedges into the ρ ring to initially isolate RNA. When the distal upstream DNA deviates from the zinc finger domain (ZBD) of RNA polymerase, NusA rotates under a cap-shaped rho subunit and then captures the RNA. After NusG separated, the clamp structure opened. As this process progressed, RNA polymerase also lost control of RNA: DNA hybrids, and at the same time, RNA polymerase lost its activity. What will happen next? The author speculates that the closing of the ρ ring may cause the termination of transcription, and eventually the hybrid will also be unlocked.

To sum up, in other words, ρ does not first bind to RNA and then help RNA to separate from DNA as stated in the textbook, but rides on the “hitchhikes” of RNA polymerase, and ρ cooperates with other proteins through structural changes make RNA polymerase inactivated, which ultimately leads to the release of RNA and the termination of transcription.

Although this process was found in Escherichia coli, the author believes that in eukaryotes, ρ functions similarly to RNA/DNA helicase Sen1 to release RNAPII from non-coding RNA. This process also requires EC intermediates to participate in an efficient termination process. Therefore, the author believes that in other species, the process of ρ-mediated RNA release is similar.