Figure 2.

Requirement of Sus1p for recruitment of TBP and RNA polymerase II to the GAL1 core promoter. (A and B) Sus1p is required for recruitment of TBP and RNA polymerase II to the GAL1 core promoter. Yeast strains were grown and crosslinked as in Figure 1B. Immunoprecipitation was carried using an anti-TBP antibody (obtained from Michael R. Green; University of Massachusetts Medical School) against TBP or 8WG16 antibody (Covance, Inc.) against the carboxy terminal domain of Rpb1p. Immunoprecipitated DNA was amplified by PCR using primer pair targeted to the GAL1 core promoter or an inactive region within chromosome V. The maximum ChIP signal was set to 100, and other ChIP signals were normalized with respect to 100. The normalized ChIP signal (represented as normalized occupancy) is plotted in the form of a histogram. (C) Transcription. Total cellular RNA was prepared from the wild type and Δsus1 strains, and mRNA levels from the GAL1 and RPS5 genes were quantitated by primer extension analysis. Yeast cells were grown as in Figure 1B. The mRNA level of wild type strain was set to 100, and mRNA level of the Δsus1 strain was normalized with respect to 100. Normalized levels of mRNA are plotted in the form of a histogram. (D) Growth analysis. Both the wild type and Δsus1 strains were initially grown in YPR up to an OD₆₀₀ of 0.1 and then switched to YPG at 30 °C. Subsequently, OD₆₀₀ was measured for next 5 hr, and plotted as a function of time.