Nowadays, a chef does not pass muster unless he or she can wield a state-of-the-art, hand-held gas torch. Equally, scientists need the right tools to further their field of research. One such tool in the field of transcription was the development, by the Roeder laboratory in 1979, and the Sharp and Gefter laboratories in 1980, of an in vitro assay for promoter-specific transcription by RNA polymerase II (pol II).
At the time, it was not clear that eukaryotic pol II could accurately initiate transcription in vitro. However, using a well-characterized adenovirus promoter, the groups were able to show selective and accurate transcription initiation in the presence of either crude cell extracts with purified pol II or whole-cell extracts alone. This assay opened up the field for the biochemical characterization of the eukaryotic transcription machinery — by adding or subtracting specific fractions, the factors required to reconstitute transcription could be identified.
Indeed, soon thereafter, using this assay, it was discovered that numerous activities were required for transcription initiation, of which at least one could recognize the TATA-box region of promoters before pol II binding. These activities were linked to a set of 'general transcription factors', which turned out to be essential for transcription initiation, and assisted pol II to recognize, bind to and initiate transcription from the core regions of many promoters. Biochemical studies of transcription systems derived from HeLa cells, rat liver, fruitfly and yeast were carried out by the Chambon, Egly, Roeder, Reinberg, Conaway, Parker, Kornberg, Greenleaf and Tjian laboratories, among others, during the late 1980s and early 1990s. It is often forgotten that proof of the existence of general transcription factors depended on their, sometimes painstaking, isolation and the reconstitution of pol II transcription with purified proteins.
In 1991, Joan and Ronald Conaway and colleagues were the first to accomplish this feat using the rat liver system, followed by Roger Kornberg and co-workers in the yeast system in 1992, and subsequently by others in the fruitfly and human systems. The Conaway team fractionated rat liver and identified five distinct enzyme fractions that were essential for specific transcription: α, βγ, δ, ε and τ. Four of these could be replaced by purified proteins from rat liver: TFIIB (α), TFIIF (βγ), TFIIE (ε) and TFIIH (δ). With the purification of the final factor, TFIIE, and having in hand purified pol II, TFIIB, TFIIF and TFIIH, as well as recombinant TATA-binding protein (TBP), it was possible to show that promoter-specific transcription could be reconstituted in vitro with purified factors. Crucially, this allowed the definition of the minimal transcriptional machinery required for promoter-specific transcription by pol II.
Incidentally, TFIID in higher eukaryotes was notoriously difficult to purify, but recombinant yeast TBP — which had been isolated and cloned by the Sharp and Guarente groups, and the Chambon group, in 1988 — could substitute for TFIID in certain circumstances.
Defining the minimal set of transcription factors required for pol-II-mediated transcription paved the way for subsequent studies of basal and activated transcription — experiments that would not have been possible without the right tools.
References
ORIGINAL RESEARCH PAPERS
Weil, P. A., Luse, D. S., Segall, J. S. & Roeder, R. G. Selective and accurate initiation of transcription at the Ad2 major late promoter in a soluble system dependent on purified RNA polymerase II and DNA. Cell 18, 469–484 (1979)
Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc. Natl Acad. Sci. USA 77, 3855–3859 (1980)
Conaway, J. W., Hanley, J. P., Pfeil Garrett, K. & Conaway, R. C. Transcription initiated by RNA polymerase II and transcription factors from liver: structure and action of transcription factors ε and τ. J. Biol. Chem. 266, 7804–7811 (1991)
Conaway, R. C., Pfeil Garrett, K., Hanley, J. P. & Conaway, J. W. Mechanism of promoter selection by RNA polymerase II: mammalian transcription factors α and βγ promote entry of polymerase into the preinitiation complex. Proc. Natl Acad. Sci. USA 88, 6205–6209 (1991)
FURTHER READING
Buratowski, S., Hahn, S., Sharp, P. A. & Guarente, L. Function of a yeast TATA element-binding protein in a mammalian transcription system. Nature 334, 37–42 (1988)
Cavallini, B. et al. A yeast activity can substitute for a HeLa cell TATA box factor. Nature 334, 77–80 (1988)
Sayre, M. H., Tschochner, H. & Kornberg, R. D. Reconstitution of transcription with five purified initiation factors and RNA polymerase II from Saccharomyces cerevisiae . J. Biol. Chem. 267, 23376–23382 (1992)
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Heinrichs, A. The right tools for the job. Nat Rev Mol Cell Biol 6 (Suppl 1), S12 (2005). https://doi.org/10.1038/nrm1801
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DOI: https://doi.org/10.1038/nrm1801