Region-specific initiation of mouse mammary tumor virus RNA synthesis by endogenous RNA polymerase II in preparations of cell nuclei

Abstract

Adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) were used to demonstrate initiation of mouse mammary tumor virus (MMTV) RNA in preparations of whole nuclei from control and glucocorticoid-treated MMTV-infected rat hepatoma tissue culture cells. RNA chains initiated in the cell-free reaction retain a thiol group at the 5' end and can be separated from thiol-free RNA chains by chromatography on mercury-Sepharose. The abundance of MMTV sequences was determined by nucleic acid hybridization with filter-bound DNA representing four different regions of the MMTV genome. About six times more MMTV RNA is initiated with GTP beta S than with ATP beta S. Most of the cell-free initiation of MMTV RNA occurs within or very near a 380-nucleotide section of the proviral long terminal repeat that is the presumptive site of transcription initiation in vivo. The sensitivity of MMTV RNA initiation and synthesis to alpha-amanitin and actinomycin D are characteristic of DNA-directed transcription by RNA polymerase II. Nuclei from glucocorticoid-treated cells initiate approximately 10 times more MMTV RNA than nuclei from control cells.