Significance The TATA box-binding protein (TBP) is highly conserved throughout eukaryotes and plays a central role in the assembly of the transcription preinitiation complex (PIC) at the promoter of genes. We show here that TBP can interact with nucleosomes containing the Widom-601 sequence at two different positions. One position comprises a TATA box-like sequence near the end of nucleosomal DNA, and the other position is GC-rich and located near the nucleosome dyad. TBP–nucleosome complexes must be resolved before PIC assembly can occur but may persist during mitosis when chromatin is condensed, possibly explaining how TBP can act as a bookmarking factor that transfers transcriptional memory.