In Vitro Transcription and Start Site Selection in Schizosaccharomyces pombe

Abstract

We have used the fission yeast Schizosaccharomyces pombe to establish both a biochemical and genetic system to study the roles of general transcription factors in transcription initiation. Extracts were prepared that faithfully transcribed S. pombe promoters and the results confirm that, in contrast to the budding yeast Saccharomyces cerevisiae, in vitro transcription in S. pombe initiates near to the TATA element. S. pombe transcription relies on upstream activation sequence elements and these can be replaced successfully with sites for binding Gal4-VP16 activators. Although it is mammalian-like in these respects, S. pombe initiation uses an unusual scanning mechanism. This directs initiation, preferentially using purines, within a narrow window approximately 25–40 base-pairs downstream from the edge of the TATA element. Genetic experiments showed that this scanning mechanism was associated with the properties of the TFIIB polypeptide. When human TFIIB was expressed in S. pombe , it was accepted by the endogenous transcription machinery and caused initiation to be restricted to the closer edge of this window, corresponding to the distance in humans. Preliminary experiments suggested that S. cerevisiae TFIIB was not accepted. The results enlarge the potential for using fission yeast to study the properties of general transcription factors such as TFIIB in choosing the sites at which transcription initiates.