Determinants of transcription initiation efficiency and start site selection by RNA polymerase I

Abstract

RNA polymerase I (Pol I) synthesizes pre-ribosomal RNA, which is essential for ribosome biogenesis. Increased Pol I activity supports rapid cell growth, a key characteristic of cancer cells. Consequently, drugs targeting Pol I in cancer are being actively investigated. The first step in pre-rRNA synthesis involves the assembly of the Pol I transcription initiation complex on the rRNA gene promoter, yet the quantitative and dynamic parameters of this process remain inadequately defined. We combined biochemical, biophysical, and molecular dynamics simulation analyses to enhance molecular models illustrating how Pol I apparatus isolated Saccharomyces cerevisiae recognizes the promoter and the transcription start site (TSS). Time-resolved binding data shows that CF relies on a two-step mechanism, consisting of the binding and isomerization steps, to recognize its binding site on the promoter. The next step, CF-mediated recruitment of the Pol I- Rrn3 complex to the promoter, was inefficient, leading to the rapid dissociation of Pol I. The selection of the TSS by Pol I is influenced by the distance to CF and the properties of downstream DNA, such as bendability. The dynamics revealed in the Pol I transcription apparatus establish a framework for comparing the functions and mechanisms of Pol I activators and inhibitors.