The general transcription factor IIB (TFIIB) plays an essential role in transcription of protein-coding genes by eukaryotic RNA polymerase II. We previously identified a yeast TFIIB mutant (R64E) that exhibited increased activity in the formation of stable TATA-binding protein-TFIIB-DNA (DB) complexes in vitro. We report here that the homologous human TFIIB mutant (R53E) also displayed increased activity in DB complex formation in vitro. Biochemical analyses revealed that the increased activity of the R64E mutant in DB complex formation was associated with an altered protease sensitivity of the protein and an enhanced interaction between the N-terminal region and the C-terminal core domain. These results suggest that the intramolecular interaction in yeast TFIIB stabilizes a productive conformation of the protein for the association with promoter-bound TATA-binding protein.