|Proofreading RNA: Structure of RNA Polymerase II's Backtracked State|
RNA polymerase II (pol II) is responsible for the production of messenger RNA, which serve as templates for the synthesis of all proteins, including key enzymes, scaffold proteins, hormones, etc. Because a low error rate during transcription is critical, pol II is very selective in nucleotide triphosphate (NTP) loading and incorporation; it also uses proofreading to improve overall transcription accuracy. During RNA transcription, pol II occasionally reverse-translocates—or backtracks—along the growing strand of RNA, correcting any mistakes that have been made. The newly created (3′) end of the RNA strand is extruded from the active center of pol II, allowing the RNA transcript to be checked and repaired.
Pol II assumes one of three major states during the transcription elongation phase. The pre-translocation state occurs when a newly added nucleotide still occupies pol II's nucleotide addition site. In the post-translocation state, the nucleotide addition site is vacant, available for the next NTP. The backtracked state occurs during reverse-translocation and is dominant during nucleotide misincorporation or when pol II runs into DNA damage or other impediments.
The structures of the pre-translocation and post-translocation states were solved in 2001 and 2004. In this research, the structure of the pol II complex in the backtracked state was solved at ALS Beamlines 5.0.2 and 8.2.2.
Using a hybrid containing one mismatched residue at the 3′ end of the RNA, researchers found the last correctly matched residue positioned within the nucleotide addition site, and the mismatched residue located at a novel site called 'P' for proofreading. The mismatched residue's interaction with pol II distorts the RNA–DNA helix, making forward transcription difficult. The enzyme's equilibrium shifts toward the backtracked state.
One of two important conclusions of this research is that pol II backtracked by one residue is stable, even reversible. In the course of backtracking, pol II stalls at this position, supporting the idea that there is equilibrium between forward and backward motion during transcription. This confirms that backtracking one residue is preferable to going back several residues, which can lead to arrest (irreversible backtracking). Recovery from arrest is only possible by cleaving the transcript and excising the misincorporated nucleotide(s).
The second conclusion is that the distorted helix that causes pol II to backtrack one residue allows for cleavage by elongation factor IIS (TFIIS) and for intrinsic cleavage (cleavage without TFIIS). However, the one-residue backtracked state is more readily cleaved in the presence of TFIIS, which rescues the complex from arrest and releases a dinucleotide. This strengthens the theory that cleavage occurs in the pol II active site, and that such cleavage is important for removal of misincorporated nucleotides.
In summary, pol II's forward movement along a DNA template is driven by NTP loading during normal transcription elongation—unless a mismatch causes the RNA–DNA helix to distort, shifting the polymerase into the backtracked state. If it remains in the backtracked state for too long, cleavage ensues. The one-residue backtracked state is a key contributor to pol II's proofreading ability, and plays an important role in increasing the fidelity of RNA polymerase.
Research conducted by D. Wang, D.A. Bushnell, X. Huang, K.D. Westover, M. Levitt, and R.D. Kornberg (Stanford University School of Medicine).
Research funding: National Institutes of Health. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.
Publication about this research: D. Wang, D.A. Bushnell, X. Huang, K.D. Westover, M. Levitt, and R.D. Kornberg, "Structural basis of transcription: Backtracked RNA polymerase II at 3.4 angstrom resolution," Science 324, 5931 (2009).