The sliding clamp model of transcription processivity, based on extensive studies of Escherichia coli RNA polymerase, suggests that formation of a stable elongation complex requires two distinct nucleic acid components: an 8-9-nt transcript-template hybrid, and a DNA duplex immediately downstream from the hybrid. Here, we address the minimal composition of the processive elongation complex in the eukaryotes by developing a method for promoter-independent assembly of functional elongation complex of S. cerevisiae RNA polymerase II from synthetic DNA and RNA oligonucleotides. We show that only one of the nucleic acid components, the 8-nt RNA:DNA hybrid, is necessary for the formation of a stable elongation complex with RNA polymerase II. The double-strand DNA upstream and downstream of the hybrid does not affect stability of the elongation complex. This finding reveals a significant difference in processivity determinants of RNA polymerase II and E. coli RNA polymerase. In addition, using the imperfect RNA:DNA hybrid disturbed by the mismatches in the RNA, we show that nontemplate DNA strand may reduce the elongation 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.

• PMID: 10692458
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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Kireeva ML, Komissarova N, Waugh DS, Kashlev M

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