Regulation of eukaryotic messenger RNA transcription is governed by DNA sequence elements that serve as binding sites for sequence-specific transcription factors. These include upstream and downstream promoter-proximal elements, enhancers, repressors, and silencers, which modulate the rate of specific initiation by RNA polymerase II. In addition, the promoter-proximal region between -45 and +30 (relative to the start of initiation) contains two highly conserved motifs, the TATA sequence at around -30 and CA at +1. Although the TATA element-binding factor TFIID has been purified and cloned from several organisms and has provided invaluable insight into the process of transcription initiation and its regulation, little is known about factors that interact at the +1 region. We have recently shown that the adeno-associated virus type 2 P5 promoter +1 region (P5 + 1 element) binds transcription factor YY1. We report here that this sequence is necessary and sufficient for accurate basal transcription. Further, partially purified YY1 can restore basal level transcription from a P5 + 1 element in a HeLa extract depleted for YY1 or a Drosophila embryo extract devoid of YY1 activity, whereas a YY1-specific antibody can block the reactivation. Finally, using electrophoretic mobility shift assay, we have identified YY1-related factors that bind to two other transcription initiators in cellular genes.