Purification of RNA polymerase II general transcription factors from rat liver

Abstract

Eukaryotic messenger RNA synthesis is a complex biochemical process requiring the concerted action of multiple “general” transcription factors (TFs) that control the activity of RNA polymerase II at both the initiation and elongation^, stages of transcription. Because the general transcription factors are present at low levels in mammalian cells, their purification is a formidable undertaking. For this reason we explored the feasibility of using rat liver as a source for purification of the general factors. Rat liver has proven to be an ideal model system for biochemical studies of transcription initiation and elongation by RNA polymerase II (Figs. 1 and 2). In our hands the yield of general transcription factors per gram of rat liver is roughly equivalent to their yield per gram of cultured HeLa cells. Moreover, we have been able to develop convenient and reproducible methods for preparation of rat liver extracts from as much as 1 kg of liver per day. Because it is both technically difficult and expensive to obtain such quantities of cultured cells on a daily basis, rat liver provides a significant logistic advantage for purification of the general transcription factors.

Fig. 1

Resolution and purification of RNA polymerase II general transcription factors from rat liver. TFIIF (rat βγ); TFIIB (rat α); TFIIH (rat δ); TFIID (rat τ); SII; TFIIE (rat ε); TFIIA; SIII (Elongin).

Fig. 2

Sodium dodecyl sulfate-polyacrylamide gel analysis of RNA polyacrylamide gel analysis of RNA polymerase II general transcription factors from rat liver. Subunits of purified general transcription factors are indicated by black circles.