Thermodynamic and kinetic studies of transcription factor IIIB
Cloutier, Timothy Edward
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This dissertation focuses on the specific transcription apparatus of the Saccharomyces cerevisiae RNA pol III system, which requires the participation of the initiation factor TFIIIB. High levels of RNA polymerase (pol) III gene transcription are achieved by facilitated recycling of the polymerase on TFIIIB-DNA complexes that are stable through multiple rounds of initiation. TFIIIB-DNA complexes in yeast comprise the TATA-binding protein TBP, the TFIIB-related factor TFIIIB70 (Brfl) and TFIIIB90 (B″ ). The high stability of the TFIIIB-DNA complex is conferred by TFIIIB90 binding to TFIIIB70-TBP-DNA (B'-DNA) complexes. This stability is thought to result from compound bends introduced in the DNA by TBP and TFIIIB90 and by protein-protein interactions that obstruct DNA dissociation. My objective has been to examine the energetic and kinetic contributions of TFIIIB90 to the assembly of TFIIIB on DNA.;Quantitative DNase I footprinting analysis shows that the free energies of formation of B'-DNA (DeltaG° = -12.10 +/- 0.12 kcal/mol) and TFIIIB-DNA (DeltaG° = -11.90 +/- 0.14 kcal/mol) complexes are equivalent whereas the half-lives of these complexes (46 +/- 3 min and 95 +/- 6 min, respectively) differ significantly. The differential stability of these isoenergetic complexes demonstrates that TFIIIB90 binding energy is used to drive conformational changes that increase the barrier to complex dissociation and kinetically trap the promoter DNA. Additional thermodynamic data reveals an interdependent cooperative relationship between TFIIIB90 and TFIIIB70 in their ability to enhance TBP's affinity for TATA DNA. Such reciprocity between these transcription factors indicates that the various association reactions are freely reversible. The binding of TBP to DNA proceeds via a multi-step reaction mechanism. Kinetic data demonstrate that TFIIIB90 and TFIIIB70 make distinct contributions to increasing TBP's association with the DNA. For example, fast-mixing pre-steady state kinetic studies reveal that TFIIIB90 enhances TBP association in the assembly of TFIIIB-DNA complexes approximately nine-fold over the binary TBP-DNA complex (71 +/- 1 x 105 and 8.5 +/- 1 x 105 M-1s-1, respectively) and threefold over the ternary B'-DNA complex (27 +/- 3 x 105 M-1s-1). Therefore, the biochemical data presented in this thesis demonstrate that TFIIIB90 works to both facilitate the assembly of the TFIIIB-DNA complex by increasing the TBP association rate and to stabilize the complex by increasing the energy barrier to dissociation.