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J Biol Chem, Vol. 275, Issue 9, 6530-6536, March 3, 2000
From the Advanced BioScience Laboratories, Inc.-Basic Research
Program, NCI-Frederick Cancer Research and Development Center, National
Institutes of Health, Frederick, Maryland 21702-1201
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.
The 8-Nucleotide-long RNA:DNA Hybrid Is a Primary Stability
Determinant of the RNA Polymerase II Elongation Complex*
*
This work was supported by the National Cancer Institute,
Department of Health and Human Services, under contract with Advanced BioScience Laboratories, Inc.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Advanced BioScience
Laboratories, Inc.-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Bldg. 539, Room 222, Frederick, MD
21702-1201. Tel.: 301-846-1798; Fax: 301-846-6988; E-mail: mkashlev@mail.ncifcrf.gov.
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