We have conducted a quantitative thermodynamic study of the effects of the TATA element and TATA-flanking sequences on the assembly of complexes containing TATA-binding protein (TBP) and the TFIIB-related factor, TFIIIB70. TBP binds to the sequence TATAAAAG in the context of the yeast U6 gene (yU6 hybrid TATA) or the adenovirus major late promoter (AdMLP) with different affinities demonstrating that the sequence context of a TATA element contributes to TBP binding. We also determined the cooperative free energies of formation of TBP.TFIIIB70.DNA complexes on the yU6 TATA element, the yU6 hybrid TATA element and a nonconsensus TATA element. The yU6 hybrid TATA displayed a moderate, less than 5-fold, increase in TBP affinity similar to the 3-fold increase observed for the AdMLP. In contrast, the nonconsensus and yU6 TATAs increased the affinity of TBP for DNA 12- and 17-fold, respectively. Since the TBP-TFIIIB70 cooperativity is greater on lower affinity TATA boxes and most polymerase III genes contain low affinity "TATA boxes," we conclude that the cooperative binding of TFIIIB70 and TBP to DNA represents an important driving force in the assembly of polymerase III-specific transcription complexes. An effect of the sequences surrounding the TATA box was also observed on TBP-TFIIIB70 cooperativity. The mechanistic implications of the thermodynamic linkage between DNA sequence and binding cooperativity are discussed.