Effects of estrogen on gene expression in the chick oviduct. IV. Initiation of RNA synthesis on DNA and chromatin

Abstract

The interaction of Escherichia coli RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6.) with chick oviduct DNA and chromatin has been studied in order to quantify the RNA polymerase binding sites and rifampicin-resistant initiation sites. The number of high affinity binding sites at saturation was calculated from the number of molecules of enzyme bound to DNA or to chromatin. The number of rifampicin-resistant initiation sites was calculated from the total quantity and the number average chain length of RNA synthesized under the conditions in which only RNA polymerase already in a stable preinitiation complex could avoid inhibition by the drug. Using an assessment of the temperature requirement for the information of this preinitiation complex, we were able to differentiate initiations at single-stranded or nicked DNA regions from initiations at double-stranded DNA regions. This method enabled us to rule out the possibility that RNA chain initiation on chromatin was due to initiation at nicked or end regions of DNA. In addition, we also demonstrated that not all RNA polymerase molecules bound to DNA or chromatin are located at a site at which RNA chain initiation can immediately begin. This methodological approach should allow investigators to monitor the individual reactions and factors involved in in vitro transcription of eukaryotic DNA and chromatin.