The 8-nucleotide-long RNA : DNA hybrid is a primary stability determinant of the RNA polymerase II elongation complex

The sliding clamp model of transcription processivity, based on extensive s tudies of Escherichia coli RNA polymerase, suggests that formation of a sta ble elongation complex requires two distinct nucleic acid components: an 8- 9-nt transcript-template hybrid, and a DNA duplex immediately downstream fr om the hybrid. Here, we address the minimal composition of the processive e longation complex in the eukaryotes by developing a method for promoter-ind ependent assembly of functional elongation complex of S. cerevisiae RNA pol ymerase II from synthetic DNA and RNA oligonucleotides, me show that only o ne of the nucleic acid components, the 8-nt RNA: DNA hybrid, is necessary f or the formation of a stable elongation complex with RNA polymerase II. The double-strand DNA upstream and downstream of the hybrid does not affect st ability of the elongation complex. This finding reveals a significant diffe rence in processivity determinants of RNA polymerase II and E. coli RNA pol ymerase. In addition, using the imperfect RNA:DNA hybrid disturbed by the m ismatches in the RNA, we show that nontemplate DNA strand may reduce the el ongation complex stability via the reduction of the RNA:DNA hybrid length. The structure of a "minimal stable" elongation complex suggests a key role of the RNA:DNA hybrid in RNA polymerase II processivity.