Specific DNA features of the RNA polymerase I core promoter element targeted by core factor

Abstract

RNA polymerase I (Pol I) is essential for ribosomal RNA (rRNA) synthesis, driving ribosome biogenesis in eukaryotes. Transcription initiation by Pol I requires core factor (CF) binding to the core element (CE) of the ribosomal DNA (rDNA) promoter. Despite structural conservation across species, significant sequence variability suggests CF recognizes DNA through structural features rather than specific sequences. We investigated CF's DNA binding preferences to elucidate the role of DNA structural properties in CE recognition. Analysis of CE sequences from 35 fungal species revealed conserved structural features, notably a rigid AT-rich patch at positions −22 to −20 and a conserved G base pair at position −24. Competition-based electrophoretic mobility shift assays (EMSA) with single base-pair substitutions showed CF tolerates mutations at many positions but is sensitive to changes in the AT-rich patch. Loss of CF binding correlated with alterations in DNA structural properties such as increased bendability, decreased curvature, widened minor groove width, and altered helix twist. In vitro SELEX experiments identified novel CE sequences preferentially bound by CF, exhibiting increased GC content, higher bendability, and decreased curvature despite lacking sequence conservation. Classification based on bendability profiles revealed CF preferentially binds bendable sequences. In vivo selection assays confirmed these findings, demonstrating consistent CF binding preferences within a cellular context. Our results indicate that CF recognizes and binds to the CE primarily through specific DNA structural features rather than nucleotide sequences. Structural properties like bendability, curvature, and minor groove width are critical determinants of CF binding, facilitating effective Pol I transcription initiation.