Formation and crystallization of yeast RNA polymerase II elongation complexes

Minimal templates were devised for the efficient generation of yeast RNA polymerase II transcription elongation complexes. A 33-base pair DNA with a 15-residue dC tail at one 3'-end supported the formation of a complex containing the polymerase paused at nucleotide 11 of the duplex region and an RNA of 14-16 residues. The same template could yield an arrested complex with the enzyme at nucleotide 13-15 and RNA of 15-17 residues. These complexes were stable for at least a week under various conditions and could be resolved by gel electrophoresis or purified by ion exchange chromatography. The purified paused complex formed crystals capable of x-ray diffraction to 3.5 A resolution. The complex remained active in the crystal and, in the presence of nucleoside triphosphates, could efficiently extend the transcript in situ.