Figure 5.

YY1-directed transcription from linear DNA containing a mismatched sequence at the initiation site. (A) Sequences of oligonucleotides with normal and altered YY1 binding sites. Interactions between YY1 and its recognition site (37) are indicated for oligonucleotide a. (B) Band-shift assay comparing the ability of YY1 to interact with its normal recognition site and modified sites containing mismatched sequences. ³²P-labeled and unlabeled competitor oligonucleotides are identified at the top of the lanes. Lanes: a^−Y, received oligonucleotide but no YY1; F, free DNA; and C, YY1–DNA complexes. (C) In vitro transcription assayed by primer extension. Transcription reactions received as template supercoiled (S) or linear (L) plasmid DNA with the AAV P5 promoter, the indicated oligonucleotide, or oligonucleotide b in a reaction lacking YY1 (b^−Y). Markers (M) were generated by chemical sequencing using the A > C reaction of oligonucleotide a; initiation sites and bands corresponding to YY1-dependent initiation events are designated with arrows. (D) In vitro transcription monitored by run off assay. Reactions received oligonucleotide a, b, or c as template. The sizes of marker DNAs are indicated in bases; specific transcripts originating from the YY1-directed initiation sites and nonspecific transcripts form the ends of the bubbles are labeled.