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Nucleic Acids Research, 1982, Vol. 10, No. 24 8311-8322
© 1982

MOLECULAR BIOLOGY

A comparison of nucleoside (ß-S) triphosphates and nucleoside ({gamma}-S) triphosphates as suitable substrates for measuring transcription initiation in preparations of cell nuclei

Lynette D. Washington and Michael R. Stallcup

Department of Biology, University of South Carolina Columbia, SC29208, USA

Received July 29, 1982. Accepted November 9, 1982.

RNA chains initiated with nucleoside (ß-S)triphosphates and ({gamma}-S)triphosphates retain the thiol groups and can be separated from thiol-free RNA by chromatography on mercury-Sepharose. Thiol-containing mouse mammary tumor virus (MMTV) RNA synthesized by preparations of nuclei from virus-infected cells was quantitated by nucleic acid filter hybridization. With ATPßS and GTPßS, region-specific initiation of MMTV RNA chains was detected in the cell free system. However, with ATP{gamma}S and GTP{gamma}S, region-specific initiation was not clearly demonstrable. The nuclear preparations can also transfer thiol groups, presumably in the form of thiophosphate, from ATP{gamma}S or GTP{gamma}S onto preexisting RNA molecules; little or no thiol-transfer occurs with the two (ß-S)-analogues. The thiophosphate transfer activity apparently interferes with the measurement of RNA chain initiation with ATP{gamma}S and GTP{gamma}S.


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