title: The 8-nucleotide-long RNA:DNA hybrid is a primary stability determinant of the RNA polymerase II elongation complex.
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.
authors.affiliation: Advanced BioScience Laboratories, Inc.-Basic Research Program, NCI-Frederick Cancer Research and Development Center, National Institutes of Health, Frederick, Maryland 21702-1201, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Kireeva
authors.forename: N
authors.initials: N
authors.lastname: Komissarova
authors.forename: D S
authors.initials: DS
authors.lastname: Waugh
authors.forename: M
authors.initials: M
authors.lastname: Kashlev
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D019921:Biotinylation; D004247:DNA / Q000378:metabolism; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D009692:Nucleic Acid Heteroduplexes; D004247:DNA; D012319:RNA Polymerase II