Ying–Yang 1 protein (YY1) supports specific, unidirectional initiation of messenger RNA production by RNA polymerase II from two adjacent start sites in the adeno-associated virus P5 promoter, a process which is independent of the TATA box-binding protein (TBP). The 2.5-Å resolution YY1-initiator element cocrystal structure reveals four zinc fingers recognizing a YY1-binding consensus sequence. Upstream of the transcription start sites protein–DNA contacts involve both strands and downstream they are virtually restricted to the template strand, permitting access to the active center of RNA polymerase II and ensuring specificity and directionality. The observed pattern of protein–DNA contacts also explains YY1 binding to a preformed transcription bubble, and YY1 binding to a DNA/RNA hybrid analog of the P5 promoter region containing a nascent RNA transcript. A model is proposed for YY1-directed, TBP-independent transcription initiation.