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

J Biol Chem. 2000 Mar 3;275(9):6530-6. doi: 10.1074/jbc.275.9.6530.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Biotinylation
  • DNA / metabolism
  • Enzyme Stability
  • Molecular Sequence Data
  • Nucleic Acid Heteroduplexes / chemistry
  • Nucleic Acid Heteroduplexes / metabolism*
  • Nucleic Acid Hybridization
  • RNA Polymerase II / metabolism*
  • Saccharomyces cerevisiae / enzymology*

Substances

  • Nucleic Acid Heteroduplexes
  • DNA
  • RNA Polymerase II