Figure 2

Simulation of the TBP regulatory network. (a) The reaction mechanism as entered into the KinTekSim program. Each numbered line represents a reaction segment governing TBP regulation and PIC assembly shown in Figure 1. The '= =' denotes forward and reverse reactions governed by 16 forward and 16 reverse "flux constants". See Additional data file 1 for definitions of terms. (b) Simulated output. The simulator was initiated with a set of values for species concentrations and flux constants governing each numbered reaction segment in (a) (in this example we used values from cluster 5 described below). The simulation was run to steady-state. The reported steady-state concentrations of the indicated protein-promoter complexes (gray bars, left graph) and RNA (black bar, left graph) are plotted as output in arbitrary units. In the right bar graph, the indicated flux constants governing the reaction segments defined in panel (a) were reduced to mimic the physiological effect of a mutation. Changes in RNA output resulting from the perturbation are plotted on a log₂ scale, as is common for expression profiling. (c) Experimental design for probing the TBP regulatory network. The left illustration is an outline of TBP with the approximate location of amino acids targeted for mutagenesis, and the interactions that are affected. The right illustration describes the strain design. Four basic strains derived from Y13.2 were constructed that contained or lacked the TAF1 TAND domain (TFIID pathway) and contained or lacked SPT3 (SAGA pathway). A plasmid expressing the indicated HA-tagged TBP mutants under the control of the GAL10 promoter was introduced into each strain, with the exception that the TFIIA-interaction mutants were introduced only into the WT and taf1(ΔTAND) strains. TBP plasmids in the spt3Δ strains utilized the 2 micron origin. Since full galactose induction requires SAGA (Spt3), it was necessary to employ the 2 micron high copy plasmid and a longer induction period in the spt3Δ strains to achieve TBP expression levels comparable to those in wild-type SPT3 strains. These procedural modifications did not alter the experimental outcomes, except to provide quantitatively more robust effects (data not shown). Evidence that the TBP mutations affect the targeted interactions is provided in section 2 of Additional data file 1.