A role for divalent cations in specifying the start site for transcription from chromatin templates in vitro

An assay that employs nucleoside 5'-O-(2-thiotriphosphates) was used to detect initiation of mouse mammary tumor virus (MMTV) RNA chains in preparations of isolated nuclei from cultured rat hepatoma cells containing stably integrated proviruses. RNA chains initiated with adenosine 5'-O-(2-thiotriphosphate), guanosine 5'-O-(2-thiotriphosphate), or uridine 5'-O-(2-thiotriphosphate) were separated from the remaining RNA by mercury-Sepharose column chromatography and analyzed for correctly initiated RNA chains with a T1 nuclease protection assay. Combined use of the thionucleotide transcription reaction with the T1 nuclease assay allowed precise localization of the transcription start sites. The majority of MMTV RNA chains were initiated with guanosine 5'-O-(2-thiotriphosphate) at a template site 133 nucleotides upstream from a PvuII site that coincides with the right end of the long terminal repeat. However, some RNA chains were also initiated with adenosine 5'-O-(2-thiotriphosphate) and uridine 5'-O-(2-thiotriphosphate) at template sites within three nucleotides of the primary guanosine start site. When Mn2+ was substituted for Mg2+ in the transcription reaction, MMTV RNA chains were initiated with approximately the same efficiency, but the start site was shifted to a position approximately 40 nucleotides downstream from the physiological start site; in the presence of Mn2+, MMTV RNA chains were initiated only with guanosine 5'-O-(2-thiotriphosphate). When the nuclei were exposed to both Mn2+ and Mg2+, transcription initiated at the manganese-dependent site. Mn2+ also caused the transcription start site for 45 S pre-rRNA to shift about 10 nucleotides upstream from the physiologically correct start site.