FIG. 5.

Requirement for either the upstream domain of the polI promoter or the UAF complex can be bypassed in vitro by overexpression of TBP. (A) Transcription extracts were made from either wild-type (WT) (RLY01) cells (lanes 1 through 6) or from wild-type cells overexpressing TBP (lanes 7 through 12). Each extract was used to transcribe templates bearing either a wild-type polI promoter (lanes 1, 4, 7, and 10), a promoter with an inactivated upstream domain (LS_up; lanes 2, 5, 8, and 11), or a promoter with an inactivated core domain (LS_core; lanes 3, 6, 9, and 12). Assays were performed at either a high (10-μg/ml) or a low (0.5-μg/ml) template concentration. Each extract was titrated to determine the amount which gave the maximum transcription with a wild-type template at 10 μg of DNA per ml. Thirty percent of the maximal amount of extract was used for each of the experiments whose results are shown. In wild-type extracts, the negative effect of inactivating the upstream domain (LS_up) can be overcome by increasing the template concentration (compare lanes 2 and 5). Inactivation of the core domain (LS_core) eliminates transcription at all template concentrations (lanes 3 and 6). Overexpression of TBP strongly stimulates transcription from a template lacking an upstream domain at a low template concentration (compare lanes 5 and 11). (B) Extracts were made from a ΔRRN5 strain (RLY07) or from a ΔRRN5 strain overexpressing TBP. Each extract was used to transcribe either a wild-type template (lanes 1 and 5), a template with a 5′ deletion to position −122 of the promoter (lanes 2 and 6), a template deleted to −42 (lanes 3 and 7), or a template deleted to −2 (lanes 4 and 8) at a 10-μg/ml template concentration. Extracts were used at 30% of the amount which gave maximal activity on a wild-type template. Overexpressing TBP stimulates transcription from promoters bearing deletions of the UAF binding region (lanes 6 and 7) to the same level seen with an intact promoter (lane 5).