Kinetic trapping of DNA by transcription factor IIIB.
High levels of RNA polymerase III gene transcription are achieved by facilitated recycling of the polymerase on transcription factor IIIB (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, and TFIIIB90. The high stability of the TFIIIB-DNA complex is conferred by TFIIIB90 binding to TFIIIB70-TBP-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. Here we present biochemical evidence that the high stability of TFIIIB-DNA complexes results from kinetic trapping of the DNA. Thermodynamic analysis shows that the free energies of formation of TFIIIB70-TBP-DNA (DeltaG degrees = -12.10 +/- 0.12 kcal/mol) and TFIIIB-DNA (DeltaG degrees = -11.90 +/- 0.14 kcal/mol) complexes are equivalent whereas a kinetic analysis shows that 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 and increase the barrier to complex dissociation.
Cloutier
T E
TE
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Librizzi
M D
MD
Mollah
A K
AK
Brenowitz
M
M
Willis
I M
IM
D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004355:Drug Stability; D005656:Fungal Proteins / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D012320:RNA Polymerase III / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D013816:Thermodynamics; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000378:metabolism*
C090956:BRF1 protein, S cerevisiae; C441028:BRF1 protein, human; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; D004247:DNA; D012320:RNA Polymerase III