For genes to be expressed, a complementary strand of RNA must be produced from a DNA template. During this process of transcription, a special class of enzyme called RNA polymerase moves along the DNA template, reading the DNA and producing an RNA complement. This process operates with amazingly high fidelity—the error rate is as low as one mistake for every 100,000 DNA base pairs transcribed—thanks in part to error correction by an RNA polymerase known as pol II, which "backtracks," or reverses, along the transcript to remove misincorporated or damaged nucleotides. A group from the Stanford University School of Medicine has solved the structure of pol II in the backtracked state, providing structural insights about a key mechanism for ensuring accurate transcription.

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.

Left: Structure of pol II elongation complex in the backtracked state of a complex with one mismatched residue at the 3′ end of the RNA. RNA and DNA are red and cyan. Ribonucleotides at +1 and +2 positions in the RNA are yellow and blue. Parts of the bridge helix and trigger loop are green and cyan. The bridge helix guides the template DNA strand into the active center and positions the DNA–RNA hybrid relative to the catalytic site. Right: Backtracked RNA is kinked toward the bridge helix and differs from the canonical form of RNA. Backbones of one-base–mismatch backtracked RNA (red, yellow, and blue) and canonical form RNA (gray) are superimposed. The superimposed two-base–mismatch structure is shown in magenta.

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).

ALSNews Vol. 304