It's one of my favorite research papers.
And I just to hop in here and write what the paper is about.
RNA polymerase is involved in the transcription of DNA into RNA across all living systems. Nucleotide addition happens in a sequence of steps. After every nucleotide is added the RNA polymerase forward translocates by 1 base pair.
The 3' end of RNA is not aligned well with the active site in case of misincorporation or other pause signals or events. This process known as backtracking inactivates transcription and the RNA is extruded through the secondary channel as the extension can't continue.
The enzyme has to be reactivated. To extend the RNA transcript the backtracked bases should be cleaved. RNA polymerases have this intrinsic property of transcript cleavage but this is very slow under physiological conditions. A set of elongation factors known as Gre in E.coli enhances this activity.
The Gre factor has 2 domains: NTD, and CTD. It protrudes the tip of its NTD into the RNA polymerase secondary channel which has several conserved residues, and the aspartic acid(41) and Glutamic acid(44) coordinates magnesium ion which activates the nucleophile(the hydroxyl group) and also complements the active site.
The paper aims to elucidate the structure of backtracked RNA in complex with Gre factor using single particle cryoelectron microscopy. They reconstitute 4 different complexes at different stages of Transcript cleavage that span the entire reaction pathway: backtracked complex, GreB bound complex before transcript cleavage and after, reactivated complex bound to rNTP and GreB.
A functional backtracked complex with three backtracked bases is created. From cryoEM data they were able to identify the residues interacting with the backtracked bases. The conformation of the backtracked bases is different from how the bases would orient themselves during elongation phase and block trigger loop(TL) folding into trigger helix(TH).
It is shown that in the presence of GreB the transcript cleavage is enhanced. The conserved residues of GreB at the N-terminal push the bases inside. The binding of GreB produces a local conformational change such that it speeds up the intrinsic transcript cleavage of RNA polymerase. Once the transcript is cleaved it again produces a conformational change that reduces the affinity between RNA polymerase and GreB. Once a nucleotide triphosphate enters the active site, RNA polymerase is reactivated. It shows the interacting residues of GreB with RNA polymerase before cleavage and how it changes once the misincorporated bases are cleaved off.
The paper beautifully demonstrates the power of CryoEM in demonstrating the mechanism of a reaction or pathway with a high resolution with the help of different stages of the complex during the reaction.
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