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Stability of Drosophila RNA polymerase II elongation complexes in vitro
Journal article   Open access   Peer reviewed

Stability of Drosophila RNA polymerase II elongation complexes in vitro

Daniel D Kephart, Nicholas F Marshall and David H Price
Molecular and cellular biology, Vol.12(5), pp.2067-2077
05/1992
DOI: 10.1128/mcb.12.5.2067-2077.1992
PMCID: PMC364378
PMID: 1373806
url
https://www.ncbi.nlm.nih.gov/pmc/articles/364378View
Open Access

Abstract

We show that nuclear extract from Drosophila Kc cells supports efficient elongation by RNA polymerase II initiated from the actin 5C promoter. The addition of 0.3% Sarkosyl, 1 mg of heparin per ml, or 250 mM KCl immediately after initiation has two effects. First, the elongation rate is reduced 80 to 90% as a result of the inhibition of elongation factors. Second, there is an increase in the amount of long runoff RNA, suggesting that there is an early block to elongation that is relieved by the disruptive reagents. Consistent with the first effect, we find that the ability of factor 5 (TFIIF) to stimulate the elongation rate is inhibited by the disruptive agents when assayed in a defined system containing pure RNA polymerase II and a dC-tailed template. The disruptive agents also inhibit the ability of DmS-II to suppress transcriptional pausing but only slightly reduce the ability of DmS-II to increase the elongation rate twofold. The pause sites encountered by RNA polymerase II after initiation at a promoter and subsequent treatment with the disruptive reagents are also recognized by pure polymerase transcribing a dC-tailed template. It has been suggested that 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits RNA polymerase II during elongation, but we find that the purine nucleoside analog has no effect on elongation complexes containing RNA over 500 nucleotides in length or on the action of factor 5 or DmS-II in the defined system.
 Cell Line Oligodeoxyribonucleotides Drosophila Molecular Sequence Data Templates, Genetic RNA Polymerase II - metabolism Promoter Regions, Genetic - drug effects Restriction Mapping Actins - genetics Transcription Factors - metabolism RNA - biosynthesis RNA - isolation & purification Animals Base Sequence Potassium Chloride - pharmacology Transcription, Genetic Sarcosine - analogs & derivatives Heparin - pharmacology Sarcosine - pharmacology

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