Optimized reaction conditions and specific inhibitors for initiation of transcription by RNA polymerase II in nuclei from cultured mammalian cells.

Abstract

An assay that employs guanosine 5'-O-(2-thiotriphosphate) was used to measure correct initiation of RNA chains in isolated cell nuclei, where chromatin structure is relatively undisturbed. RNA chains initiated with guanosine 5'-O-(2-thiotriphosphate) were separated from the remaining RNA by mercury-Sepharose column chromatography and analyzed for correctly initiated mouse mammary tumor virus RNA with a T1 nuclease protection assay. The monovalent cation concentration dependence for initiation in isolated nuclei was similar to that previously observed for initiation from naked DNA templates (optimum near 90 mM) but different from that for elongation of nascent RNA chains. However, in contrast to the systems that employ naked DNA templates, initiation efficiency in the nuclear system was relatively unaffected by moderate changes in pH (6.7-8.3), temperature (25-37 degrees C), and magnesium ion concentration (1-9 mM). The optimized assay was used to assess the inhibitory activity of several compounds that have been reported to be specific inhibitors of transcription initiation on naked DNA templates. Both Sarkosyl and heparin were effective inhibitors of specific initiation by RNA polymerases I and II in isolated nuclei without inhibiting elongation of nascent chains, but 5,6-dichlorobenzimidazole riboside was relatively ineffective as a specific inhibitor of initiation by RNA polymerase II.