Structural Basis of Transcription: Backtracked RNA Polymerase II at 3.4 Angstrom Resolution

Stepping Back to Go Forward Insight into the mechanism of transcription has come from crystal structures of actively transcribing RNA polymerase II complexes in the pre- and posttranslocation states. RNA polymerase also backtracks on the DNA template. Backtracking by only a few residues is reversible, but longer backtracking leads to arrest that is relieved by cleavage of the transcript by the transcription elongation factor SII (TFIIS). Now Wang et al. (p. 1203) report x-ray structures of backtracked ternary complexes and of a backtracked complex bound to a noncleaving mutant of TFIIS. The structures show a defined one-residue, backtracked state supporting the idea that RNA polymerase oscillates between backward and forward motion during active transcription. Mismatched residues disfavor forward translocation, increasing the lifetime of the backtracked state and facilitating cleavage by TFIIS. Thus, TFIIS-induced cleavage is likely to provide an important proofreading function during transcription. A backtracked RNA polymerase II reveals how the enzyme proofreads the RNA transcript. Transcribing RNA polymerases oscillate between three stable states, two of which, pre- and posttranslocated, were previously subjected to x-ray crystal structure determination. We report here the crystal structure of RNA polymerase II in the third state, the reverse translocated, or “backtracked” state. The defining feature of the backtracked structure is a binding site for the first backtracked nucleotide. This binding site is occupied in case of nucleotide misincorporation in the RNA or damage to the DNA, and is termed the “P” site because it supports proofreading. The predominant mechanism of proofreading is the excision of a dinucleotide in the presence of the elongation factor SII (TFIIS). Structure determination of a cocrystal with TFIIS reveals a rearrangement whereby cleavage of the RNA may take place.