1. Interplay of TBP Inhibitors in Global Transcriptional Control
Carmelata Chitikila, Kathryn L. Huisinga, Jordan D. Irvin, Andrew D. Basehoar, B.Franklin Pugh 
Molecular Cell 
Volume 10, Issue 4, Pages (October 2002)
DOI: /S (02)

2. Figure 1 Interaction of TBP with Regulatory Factors
(A) Structures of TBP interactions relevant to this study. Shown is the core of yeast TBP interacting with itself (Chasman et al., 1993; Nikolov et al., 1992), the Drosophila TAND I domain (Liu et al., 1998), and TATA DNA plus human NC2 (Kamada et al., 2001). The TFIIA•TBP•TATA•TFIIB structure is a composite of two structures (Nikolov et al., 1995; Tan et al., 1996). All views are from the same vantage point: upstream of the TATA box looking downstream. Selected amino acids are shown as stick diagrams. The relative affinity of each negative regulator (shown in red) for the relevant TBP mutants is shown below each diagram. Those in parentheses are not significantly different from each other.
(B) TBP's interaction surface with the yeast TAF1 TAND domain. Purified recombinant GST-scTAF1 (10–88) (denoted as TAND), GST-scTAF1(10–88, D66K), or GST-scTAF1(10–88, F23K, D66K) were immobilized on glutathione agarose in the presence of purified his-tagged TBP mutants, as indicated. The resins were washed, and proteins were analyzed for TBP and GST by SDS-PAGE and immunoblotting. 15% of the input material was loaded where indicated. Relative pull down represents the average of at least three repeats.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2
TBPEB Mutants in Combination with ΔTAND Display Dominant Synthetic Toxicity
Cultures ([A], wild-type; [B], ΔTAND), described in Figure 3, were spotted onto solid media containing 2% galactose and incubated at 30°C for several days. “Dominant synthetic toxicity” indicates that the phenotype was observed in the presence of wild-type TBP and that the growth phenotypes were severe in the ΔTAND strain but minor in the isogenic wild-type strain. The term “toxicity” is used since the cells do not die, but do stop growing. Growth was restored when the arrested mutants were transferred to media containing glucose, and it was again inhibited upon plating on galactose. Similar synthetic growth defects have been observed for several mild TBP mutants that support cell viability in the absence of wild-type TBP (Kobayashi et al., 2001).
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3
Microarray Analysis of TBP Mutants in Wild-Type and ΔTAND Strains
(A) Strains harbored either wild-type or TAF1(ΔTAND), and the indicated HA-tagged TBP derivatives under control of the GAL10 promoter. TBP expression was induced for 45 min, and equivalent numbers of cells were analyzed for TBP by immunoblotting (Jackson-Fisher et al., 1999). Purified recombinant his-TBP standards are shown.
(B) Cluster analysis of gene expression profiles. Cluster and Treeview (Eisen et al., 1998) were used to cluster 2358 significant changes in gene expression (defined in Experimental Procedures) initially into five clusters, using the K-means algorithm. Five clusters were initially chosen since clusters greater than five were visually redundant. Upon subsequent analysis, it became evident that two of the clusters represented similar mechanisms and so were merged to form group 1. Group 1 was then sorted by the values in the F182V column. Each column represents gene expression changes in the strain designated above each column. Names are colored to signify related mutations. Strains indicated by ΔTAND contained a deletion of the TAF1 TAND domain, while the remainder were isogenic wild-types. Each row corresponds to an expression ratio for a single gene (red = increased expression, green = decreased expression, black = no change, gray = missing data). The intensity of color correlates to the magnitude of change. The collection of columns were subjected to hierarchical clustering using Cluster and Treeview, and the resulting dendrogram is shown above the list of mutants.
Several experiments provide a frame of reference. (1) Two independent reference versus reference data sets (null TBP in a wild-type TAF1 strain, columns 18 and 19) are indicative of no change. (2) To assess the full reproducibility of the experiments, the V161R experiments (in both wild-type TAF1 and ΔTAND strains) were repeated approximately a year apart by different persons (V161RK versus V161RL, columns 4, 5, 11, and 12). Of the typically >5700 genes that passed filtering criteria 1 (see Experimental Procedures), correlation coefficients of 0.9 and 0.8 for wild-type and ΔTAND, respectively, were obtained, indicating a high degree of reproducibility.
(C) Dendrograms derived from hierarchical clustering of mutants in groups 1 and 4. Portions of the dendrogram that encompass the TBPEB arginine mutants are boxed in yellow. “Δ” indicates ΔTAND.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4
Dependency of Selected Groups of Genes on the TAF1 TAND Domain
For the indicated groups of genes, log2 ratios of fold changes in gene expression for the indicated mutants in the ΔTAND strain were plotted as a function of the same mutants in the corresponding wild-type strain. Two groups were plotted in each panel. TAND effects are reflected as deviations of the data points from the red diagonal.
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5 Expression Level of Various Gene Groups
Fold changes in gene expression (log2 ratio) for representative mutants in each indicated group of genes were plotted as a function of log10 expression intensity in the reference state (null TBP in a wild-type TAF1 strain). Intensities represent an average from 12 reference hybridizations. The entire nullL dataset is plotted in black to provide a frame of reference for the distribution of gene expression intensities. Group 1, represented by the V161RK mutant in the wild-type TAF1 strain, is plotted in the lower half of the panel (green). Group 4, represented by the V161RK mutant in the ΔTAND strain, is plotted in the upper half of the panel (red). Plots of other groups can be found in Supplemental Figure S6 at
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6 Subtelomeric Frequency Profile of Group 3 and 4 Genes
Shown is a composite profile of all 32 subtelomeric regions. The frequencies of nonrepetitive genes that increased in expression in a 50 gene window, tiled every 10 genes, were plotted as a function of their average distance from the telomere (Wyrick et al., 1999). Group 3 is shown in blue and group 4 in red. Also plotted (open circles) is the percentage of genes in the same 50 gene window that are in the lowest tenth percentile of genome-wide expression levels.
Molecular Cell  , DOI: ( /S (02) )

8. Figure 7
Models for the Interplay of TBP Effectors in Regulating the Four Groups of Genes Identified in This Study
Positively acting functions are shown in green, and negatively acting functions are shown in red. The thickness of the black equilibrium arrows reflects the tendency of one interaction to dominate over another.
Molecular Cell  , DOI: ( /S (02) )