What question did this study set out to answer?

To purify and characterize DNA-dependent RNA polymerase II from human cell lines.

June 1, 1973Journal of Biological Chemistry159 citationsOpen Access

Mammalian Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerases

BSBill SugdenUniversity of Wisconsin–Madison WKWalter KellerUniversity of Graz

Key Points

To purify and characterize DNA-dependent RNA polymerase II from human cell lines.
Enzyme purification from HeLa and KB cell lines was performed.
Molecular weights of the enzyme and its subunits were determined using sodium dodecyl sulfate polyacrylamide gels.
The influence of divalent cation concentration on enzyme activity was assessed.
RNA polymerase II has a molecular weight of approximately 500,000, with subunits of 220,000, 140,000, and 35,000 daltons.
A preference for single-stranded DNA over double-stranded DNA as a template was observed.
Partial purification of stimulatory factors SF-A and SF-B, which enhance RNA polymerase II activity in the presence of double-stranded DNA, was successful.

Abstract

Abstract A procedure is described for the purification of DNA-dependent RNA polymerase II of the human tissue culture cell lines HeLa and KB. The enzyme has a molecular weight of approximately 500,000 and in sodium dodecyl sulfate polyacrylamide gels invariably shows 3 subunits with molecular weights of 220,000, 140,000, and 35,000. In addition there perhaps is a fourth subunit of about 25,000 daltons or less in mass and a subunit of 170,000 daltons occurring in variable amounts. RNA polymerase II has a strong preference for single-stranded over double-stranded DNA as template and is most active in the presence of 1.5 to 3 mm Mn++ as divalent cation. We also describe the partial purification of two stimulatory factors (SF-A and SF-B) which specifically stimulate the activity of RNA polymerase II in the presence of double-stranded DNA as template.

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