Abstract
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.
Highlights
An assay that employsnucleoside5’-0-(2-thiotriof integrated mouse mammary tumor virus (MMTV)’ genes phosphates) was used todetectinitiation of mouse is stimulated by glucocorticoidhormones (Ringold, 1983)and mammary tumor virus(MMTV) RNAchains in prepa- provides a convenient model system for studying rations of isolated nuclei from cultured rat hepatoma regulation of gene expression
A complete understanding of the mechanism for transcriptional stimulation by RNA chains were initiated with guanosine 5‘-0-(2- the bound receptor-hormone complexes willrequire the estabthiotriphosphate) at a template site 133 nucleotides lishment of cell-free systems that allow reconstruction of the upstream from a PvuII site that coincides with the hormone response in vitro
As that 45 S pre-rRNA chains are initiated by RNA polymerase before in the presence of Mn2+,MMTV RNA synthesis was I at approximatelythe correct site
Summary
1-2 nucleotides on the assignment of the transcription start site. MMTV RNA chains initiated in our nuclear transcription system have the same 5’-ends (+3 nucleotides) as MMTV RNA chains from infected cells (Zhang-Keck and Stallcup, 1988). Thiol-containing RNA was isolated by Initiation of Specific RNA Species in the Cell-free Reaction-Nuclei mercury-Sepharose column chromatography, hybridized with from the MMTV-infected rat hepatoma line, M1.54, were prepared the unlabeled SS7.95 single-stranded MMTV DNA probe as described previously (Stallcup and Washington, 1983), after the cells weregrown for 30 min in growth medium containing 1 FM dexamethasone. In theregion of the polyacrylamide gel containing the 268nucleotide band, a 1-mm difference in migration represented nuclease mapping of MMTV RNA or 45 S pre-rRNA initiated with about 5 nucleotides; the width of the MMTV-specific band the thionucleotides were described previously The lengths of of these length determinations, a293-nucleotide RNA species the protected fragments were determined by comparison of their of knownsequence (see “Experimental Procedures”)was ana-
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