Chicken embryo extracts contain a factor that preferentially blocks the accumulation of RNA polymerase II transcripts in a cell-free system

Tyagi, J. S. ; Merlino, G. T. ; de Crombrugghe, B. ; Pastan, I. (1982) Chicken embryo extracts contain a factor that preferentially blocks the accumulation of RNA polymerase II transcripts in a cell-free system Journal of Biological Chemistry, 257 . pp. 13001-13008. ISSN 0021-9258

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Official URL: http://www.jbc.org/content/257/21/13001.abstract

Abstract

Chick embryos, chick embryo fibroblasts, and Rous sarcoma virus-transformed chick embryo fibroblasts contain a factor that preferentially blocks the accumulation of DNA-directed RNA polymerase II transcripts. The factor was detected by inhibition of transcription in a cell-free assay system utilizing partially purified RNA polymerase II from calf thymus, soluble factors from HeLa cells, and a purified DNA template. At low concentrations, it specifically prevents the accumulation of RNA polymerase II transcripts; at higher concentrations, it blocks the accumulation of other transcripts. The factor has been partially purified by sequential chromatography on BioRex 70, DNA-cellulose, Bio-Gel P-6, and HPX-87 from extracts of chicken embryos. The activity was resistant to treatment with trypsin, pronase, or micrococcal nuclease. A partial characterization of the molecule indicates that (i) it has an apparent molecular mass of about 200-300 daltons, (ii) it is stable at pH 2 and pH 12 and to heating at 100 ° C, (iii) it is not extractable by ether or chloroform:methanol, (2:1, v/v), and (iv) it is labile to heating at 800 ° C. These data suggest that it is a small, hydrolphilic compound probably organic in nature. The factor is active in a transcription assay utilizing either the Rous sarcoma virus Long Terminal Repeat promoter or the chick α2 (Type I) collagen-promoter as DNA templates. The accumulation of promoter-specific transcripts is blocked in a cell-free assay utilizing either Rous sarcoma virus-chick embryo fibroblast extracts or HeLa S-100 factors and calf thymus RNA polymerase II. In the absence of S-100, the factor does not appreciably affect the accumulation of randomly initiated transcripts produced by calf thymus RNA polymerase II on a DNA template; this result indicates the factor interacts directly or indirectly with some component(s) of HeLa S-100 to prevent the accumulation of RNA.

Item Type:Article
Source:Copyright of this article belongs to The American Society for Biochemistry and Molecular Biology.
ID Code:57533
Deposited On:27 Aug 2011 12:16
Last Modified:27 Aug 2011 12:16

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