Mechanistic studies of RNA polymerase II species-specific transcription initiation patterns
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The basal eukaryotic transcription machinery for protein coding genes is highly conserved from yeast to high eukaryotes. However, while human cells usually initiate at a single transcription start site approximately 30 bp downstream of a TATA element, Schizosaccharomyces pombe typically initiates at multiple sites 30-70 bp, and Saccharomyces cerevisiae 40 to 200 bp downstream of the TATA. The determinant factor(s) for the species specific initiation and the underlying mechanisms for the multiple far downstream start site utilization in yeast are not well understood. By swapping the highly purified transcription factors between S. pombe and S. cerevisiae reconstituted transcription systems, we confirmed previous observations that RNA polymerase II and/or the general transcription factor TFIIB determine the species-specific start site utilization patterns. Further genetic and biochemical assays of TFIIB chimeras indicated that RNAPII, but not TFIIB as previously proposed, determines the distinct initiation patterns not only between the two yeast systems but also between human and yeast systems. Bubble template initiation assays showed that there is an inverse correlation between the amount of negative charge in the TFIIB B-fingertip and the efficiency of the first phosphodiester bond formation. Moreover, biochemical studies indicate that multiple initiation steps, including first phosphodiester bond formation, and RNA:DNA hybrid stability determined initiation-to-elongation transition, could be modulated to regulate the far downstream start sites utilization in S. cerevisiae . A model for multiple far downstream transcription start sites formation in S. cerevisiae is proposed.