DNA-binding factors assemble in a sequence-specific manner on the maize mitochondrial atpA promoter

The maize mitochondrial atpA promoter has been well-characterized using in vitro transcription. The functional elements of this promoter comprise a central domain extending from –7 to +5 relative to the transcription start site, and an upstream domain of 1–3 bp that is purine-rich and centered around positions –11 to –12. As a first step in characterizing the transcriptional machinery, exonuclease-III mapping (toeprinting) was used to map the borders of DNA-protein interactions using either a 107-bp wild-type template or transcriptionally-inactive templates containing linker-scanning mutations. These experiments revealed that, with a wild-type promoter, protein factors occupy as much as 36 bp, from positions –20 to +16 relative to the transcription initiation site. Protein-binding patterns were altered when the linker-scanning mutants were used, suggesting that either the number or conformation of DNA-binding proteins could account for their inability to promote transcription initiation.