FIG. 5.
An upstream TATA is required to program a functional Pol III preinitiation complex on an A box- and B box-containing tRNA gene in S. pombe. Transcription complexes were monitored using template exclusion assays performed in parallel in S. cerevisiae (top) and S. pombe (bottom) in vitro transcription systems. The tRNA genes used for Fig. 2B were tested for the ability to form stable transcription complexes. After incubation of the first template (the order of addition is indicated above the lanes) with transcription buffer (previously determined to be optimal at 20 min), nucleoside triphosphates and [α-32P]GTP were added along with the second template (indicated above the lanes), and transcription was allowed to proceed. The reaction was stopped, and RNA was extracted and separated on 6% polyacrylamide–8 M urea. The templates were as follows: A, sc-tRNASer −TATA; B, sc-tRNASer +TATA; C, sp-tRNASer −TATA; D, sp-tRNASer +TATA; E, pJK148 (empty vector); F, sp-tRNASer-3T +TATA. The arrows point to the different transcripts synthesized.