Abstract
Factor 2 was previously identified in Drosophila Kc cell nuclear extract (KcN) as an activity suppressing the appearance of long transcripts (Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1987) J. Biol. Chem. 262, 3244-3255). A 154-kDa protein with factor 2 activity was purified to apparent homogeneity from KcN. An immobilized template assay indicated that factor 2 caused the release of transcripts by RNA polymerase II in an ATP-dependent manner. Some early elongation complexes were resistant to factor 2 action but became sensitive after treatment with 1 M KCl. In the absence of factor 2, transcription complexes still exhibited a low degree of processivity suggesting that factor 2 was only partially responsible for abortive elongation.
Highlights
The study of eucaryotic gene expression is aided by the application of procaryotic paradigms
The model states that all RNA polymerase II molecules that initiate from a promoter are destined to produce only short transcripts due to the action of negative transcription elongation factors (NTEF)
We purified Drosophila factor 2 and determined that it caused the release of the RNA component of RNA polymerase II elongation complexes in an ATP-dependent manner
Summary
Chromatography and Fractionation—General chromatography procedures were as described by Price et al [16]. Purification of Factor 2—Factor 2 was purified from Drosophila KcN using an in vitro transcription assay (see below). Factor 2 eluted from 180 to 220 mM KCl, and these fractions were pooled and directly loaded on a 10-ml ceramic hydroxyapatite column equilibrated with 50 mM HGPEDP. The fractions (220 –350 mM phosphate) containing factor 2 activity were pooled and dialyzed versus HGEDP until the phosphate concentration was 100 mM and loaded on a 1-ml Mono S column. The material bound to the Mono S column was eluted with a 110 – 600 mM KCl gradient. Reactions contained the following: 20 mM HEPES, 5 mM MgCl2, 600 M of each ATP, GTP, UTP, 30 M CTP, 60 mM KCl, 3 Ci of [␣-32P]CTP, 6 g/ml DNA template, and partially purified factors that supported transcription initiation and elongation.
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