The crystal structure of the complete 12 subunit RNA polymerase (pol) II bound to a transcription bubble and product RNA reveals incoming template and nontemplate DNA, a seven base pair DNA/RNA hybrid, and three nucleotides each of separating DNA and RNA. The complex adopts the posttranslocation state and accommodates a cocrystallized nucleoside triphosphate (NTP) substrate. The NTP binds in the active site pore at a position to interact with a DNA template base. Residues surrounding the NTP are conserved in all cellular RNA polymerases, suggesting a universal mechanism of NTP selection and incorporation. DNA-DNA and DNA-RNA strand separation may be explained by pol II-induced duplex distortions. Four protein loops partition the active center cleft, contribute to embedding the hybrid, prevent strand reassociation, and create an RNA exit tunnel. Binding of the elongation factor TFIIS realigns RNA in the active center, possibly converting the elongation complex to an alternative state less prone to stalling.

Preparation of yeast proteinsEndogenous S. cerevisiae ten-subunit Pol II core enzyme was prepared as described 1 . An E. coli expression vector was derived from pET21b (Novagen) for the coexpression of the translational fusion of S. cerevisiae Rpb4:20 glycine linker:TFIIB and Rpb7:His 6 under the control of separate T7 promoters. Details of the vector design are available on request. Following expression in E. coli, cells were lysed by sonication in buffer A (50 mM Tris, 150 mM NaCl; pH 7.5, 0.3 mg/L leupeptin, 1.4 mg/L pepstatin A, 0.17 g/L PMSF, 0.33 g/L benzamidine and 10 mM β -mercaptoethanol). The lysate was cleared by centrifugation and applied to a Ni-NTA agarose column (Qiagen). The column was washed with buffer A containing 2 M NaCl, and the protein was eluted with a gradient of 10 mM to 200 mM imidazole in buffer A containing 150 mM NaCl. Peak fractions were diluted twofold and loaded onto a Mono-S cation exchange column (Amersham) equilibrated with buffer A containing 100 mM NaCl. The fusion protein was eluted over a total of 15 column volumes with a gradient of 0.1-1 M NaCl in buffer A. Peak fractions were concentrated and applied to a Superose 6 gel filtration column (Amersham) equilibrated with buffer B (5 mM HEPES pH 7.25, 40 mM ammonium sulfate, 10 μM ZnCl 2 , 10 mM DTT). Peak fractions were concentrated, shock-frozen in liquid nitrogen, and stored at −80°C. The TBP core domain (S. cerevisiae residues 61-240) expression vector was a generous gift from Dr. Sean Juo. Expression and purification of the yeast TBP core domain was as described 2 except that Superose 12 size exclusion chromatography was performed with buffer B. Peak fractions were concentrated, shock-frozen in liquid nitrogen, and stored at −80°C. 10-subunit Pol II was incubated with two molar equivalents of nucleic acid scaffold (Template, 5'-cgacacagcatcaaatgcacgatgtaacttttataggcgcccaacc;Nontemplate, 5'-ggttgggcgcctataaaagttacatcgtgcaaaatcgttatgagaa; RNA, 5'-gctgtgtcg) as described 3 and 2.5 molar equivalents of TBP. After incubation for 20 minutes at 20°C 3-5 molar equivalents of TFIIB-Rpb4/7 fusion protein were added. After incubation for 20 min. at 20°C, the complex was purified on a Superose 6 size exclusion column (Amersham). Fractions corresponding to the complex were pooled and concentrated to 4 mg/ml.Crystallization, data collection, and structure determination Crystals were grown at 20 °C using the hanging drop vapor diffusion method by mixing 1.5 µl of sample solution with 1.5 µl of reservoir solution (800 mM sodium ammonium tartrate, 100 mM HEPES pH 7.5, 5 mM DTT). Crystals were transferred stepwise to mother solution containing additionally 0-22% glycerol over 8 h, slowly cooled down to 8 °C, incubated for another 24 h, and plunged into liquid nitrogen. Diffraction data were collected in 0.75° increments at the protein crystallography beamline ID 29 at ESRF. Diffraction data were processed with XDS and scaled with XSCALE 4 . The structure was solved by molecular replacement with PHASER 5 using the first 12-subunit Pol II ...