Marking the start site of RNA polymerase III transcription: the role of constraint, compaction and continuity of the transcribed DNA strand

The effects of breaks in the individual strands of an RNA polymerase III promoter on initiation of transcription have been examined. Single breaks have been introduced at 2 bp intervals in a 24 bp segment that spans the transcriptional start site of the U6 snRNA gene promoter. Their effects on transcription are asymmetrically distributed: transcribed (template) strand breaks downstream of bp -14 (relative to the normal start as +1) systematically shift the start site, evidently by disrupting the normal mechanism that measures distance from DNA-bound TBP. Breaks placed close to the normal start site very strongly inhibit transcription. Breaks in the non-transcribed strand generate only minor effects on transcription. A structure-based model interprets these observations and explains how the transcribed strand is used to locate the transcriptional start site.