RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells.

Abstract

By using a protein-DNA cross-linking method (D. S. Gilmour and J. T. Lis, Mol. Cell. Biol. 5:2009Biol. 5: -2018Biol. 5: , 1985, we examined the in vivo distribution of RNA polymerase II on the hsp7O heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp7O gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min U irradiation of chilled cells with mercury vapor lamps or by a 40-ps irradiation of cells with a high-energy xenon flash lamp.We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation.The Drosophila melanogaster hsp7O promoter region is structurally complex even in noninduced cells. Nucleasehypersensitive regions include an interval upstream of the transcription start, as well as an interval immediately downstream of the transcription start, which has a peak of sensitivity at position +42 (8, 32, 33). These features indicate the absence of standard nucleosomes and suggest the presence of nonhistone proteins (10, 11). Wu (33) discovered that a barrier to exonuclease III digestion exists in the -40 to -12 interval of the hsp7O promoter in the nuclei of noninduced cells. This barrier may be the result of a TATA box-binding protein, which has been partially purified and characterized by in vitro binding assays (25). Inspection of the pattern of exonuclease digestion (33) reveals another barrier 65 base pairs downstream of the transcription initiation site. Thus, the start of the hsp7O transcription unit may also be associated with protein. In the present paper, we discuss evidence obtained with a protein-DNA cross-linking method (13-15) which leads us to conclude that RNA polymerase II is part of this promoter complex in noninduced cells. MATERIALS AND METHODSThe protein-DNA cross-linking method was executed as previously described by Gilmour and Lis (14) for the experiments presented in Fig. 1 and 2. Antiserum against the 215,000-dalton subunit of RNA polymerase 11 (30) was used to recover RNA polymerase II-DNA adducts, and antiserum against Escherichia coli RNA polymerase (13) was used to assess nonspecific background. Experiments in Fig. 3 and 4 had the following modifications. Ten-milliliter portions of Schneider line 2 cells (26) were transferred from a spinner flask to a rectangular, siliconized glass dish (6 by 8 cm) and irradiated for either four flashes separated by 1-s intervals or a single flash from a Xenon lamp (model N734; Xenon Corp.). For the experiment with four flashes, the lamp was positioned 10 cm above the cells, whereas for the single-flash Xenon model 457 micropulser set at 7,000 V. A trigger wi...

RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo

“…3A). Second, the Pol II peaks map an average of ~50 bp downstream of the transcription start site, consistent with the location of stalled Pol II at heat shock genes 4,5,22 (Fig. 3B).…”

“…3C). Finally, we used permanganate footprint assays as an independent method to confirm stalled Pol II at selected genes 5,14 . For example, the rho gene displays clear permanganate sensitivity downstream of the transcription start site (+36 bp), consistent with the Pol II stalling profile seen in Toll 10b mutants ( Fig.…”

Nucleosome organization in the Drosophila genome

“…The location of the +1 nucleosome at the 5′ end of genes is striking because its upstream border resides at approximately +62 (relative to the TSS), which is near where Pol II pauses during the transcription cycle 3,[28][29][30][31][32] . To examine the potential linkage between Pol II pausing and nucleosome positions, we first determined the genome-wide location of Pol II in embryos at 1,956 putatively paused genes (Fig.…”

Imaging Drosophila gene activation and polymerase pausing in vivo

“…An early and surprising finding, seen initially by UV-ChIP 21 , was that Drosophila heat shock genes had a polymerase associated with Displayed is a small segment of fixed chromosome 3 before (top) and after (bottom) heat shock. Chromosomes are stained for DNA (Hoeschst dye; blue) and for Pol II (green) 29 .…”