| High mobility protein-1 (HMGB-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 HMGB-1/TBP/TATA complex, which is proposed to inhibit the assembly of the preinitiation complex. Using electrophoretic mobility shift assays, we show that the acidic C-terminal domain of HMGB-1 and the N-terminus of human TBP are the domains that are essential for the formation of a stable HMGB-1/TBP/TATA complex. HMGB-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 in the dissociation-rate constant. In support of the binding target of HMGB-1 being the N-terminus of hTBP, the N-terminus polypeptide of human TBP competes with and inhibits HMGB-1/TBP/TATA complex formation. Deletions of the N-terminus of human TBP were used to map the region(s) at which HMG-1 binds. These findings indicate that interaction of HMGB-1 with the Q-tract (55--95) in hTBP is primarily responsible for stable complex formation. In addition, the monoclonal antibody, MAb1C2, specific to the Q-tract, and HMGB-1 compete for the same site in the N-terminus of TBP. Furthermore, calf thymus HMGB-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.; Our studies with HMGB proteins were extended to its interaction with the regulatory protein, human estrogen receptor alpha (ERalpha). HMGB-1 was shown to promote transcription by increasing the sequence-specific binding affinity of a number of activators for their cognate recognition sequences and this in turn enhanced the level of transcription. As a result, HMGB-1/-2 was also classified as coactivators (of transcription). (Abstract shortened by UMI.)... |
