High mobility protein-1 (HMG-1) has been shown to regulate transcription by RNA polymerase II. In the context that it acts as a transcriptional repressor, it binds to the TATA-binding protein (TBP) to form the HMG-1/ TBP/TATA complex, which is proposed to inhibit the assembly of the preinitiation complex. By using electrophoretic mobility shift assays, we show that the acidic C-terminal domain of HMG-1 and the N terminus of human TBP are the domains that are essential for the formation of a stable HMG-1/TBP/TATA complex. HMG-1 binding increases the affinity of TBP for the TATA element by 20-fold, which is reflected in a significant stimulation of the rate of TBP binding, with little effect on the dissociation rate constant. In support of the binding target of HMG-1 being the N terminus of hTBP, the Nterminal polypeptide of human TBP competes with and inhibits HMG-1/TBP/TATA complex formation. Deletion of segments of the N terminus of human TBP was used to map the region(s) where HMG-1 binds. These findings indicate that interaction of HMG-1 with the Q-tract (amino acids 55-95) in hTBP is primarily responsible for stable complex formation. In addition, HMG-1 and the monoclonal antibody, 1C2, specific to the Q-tract, compete for the same site. Furthermore, calf thymus HMG-1 forms a stable complex with the TBP/TATA complex that contains TBP from either human or Drosophila but not yeast. This is again consistent with the importance of the Q-tract for this stable interaction and shows that the interaction extends over many species but does not include yeast TBP.The TATA-binding protein is a universal transcription factor that is essential for eukaryotic transcription by all three RNA polymerases (1-4). For RNA polymerase II transcription, the regulation of TBP 1 binding to the TATA element is considered a principal determinant in promoter activity and therefore a primary target for regulatory factors. TBP can be considered modular in nature, with its highly conserved C terminus being necessary and sufficient for both binding to the TATA box and basal level transcription (5, 6). In addition all activators and repressors that bind to human TBP (hTBP) are reported to bind to the C terminus (2-4). On the other hand, the interactions of regulatory factors with the 159-residue N terminus in hTBP appear much more limited, and its role in transcriptional regulation is not understood. In the only case that is characterized, it was shown that the N terminus down-regulates hTBP binding to the U6 TATA box, mediates cooperative binding with SNAPc to the U6 promoter, and facilitates an enhanced level of RNA polymerase III transcription of the U6 gene (7,8). In addition, a monoclonal antibody specific for the Q-tract of the N terminus of hTBP was shown to inhibit selectively in vitro transcription from TATA-containing, but not TATA-less, promoters that were transcribed by RNA pol II or III. This antibody interaction did not affect TBP binding to the TATA box or inhibit the formation of the TFIIA/TFIIB/TBP/TATA complex, which sugges...