Abstract
Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a prerequisite for the biosynthesis of ribosomes in eukaryotes. Compared to Pols II and III, the mechanisms underlying promoter recognition, initiation complex formation and DNA melting by Pol I substantially diverge. Here, we report the high-resolution cryo-EM reconstruction of a Pol I early initiation intermediate assembled on a double-stranded promoter scaffold that prevents the establishment of downstream DNA contacts. Our analyses demonstrate how efficient promoter-backbone interaction is achieved by combined re-arrangements of flexible regions in the ‘core factor’ subunits Rrn7 and Rrn11. Furthermore, structure-function analysis illustrates how destabilization of the melted DNA region correlates with contraction of the polymerase cleft upon transcription activation, thereby combining promoter recruitment with DNA-melting. This suggests that molecular mechanisms and structural features of Pol I initiation have co-evolved to support the efficient melting, initial transcription and promoter clearance required for high-level rRNA synthesis.
Highlights
Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a prerequisite for the biosynthesis of ribosomes in eukaryotes
Pol I could be recruited to a upstream activating factor (UAF)/TPB/Net1/core factor (CF)-bound promoter scaffold lacking sequence stretches required for forming extended downstream contacts with the jaw- and clamp-head domains of the polymerase
The polymerase is recruited to its double-stranded DNA (dsDNA) promoter but cannot complete the melting process due to a lack of fixated downstream DNA
Summary
Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a prerequisite for the biosynthesis of ribosomes in eukaryotes. Previous structural analyses of Pol I initiation complexes by us and others relied on an artificially stabilized, mismatched bubble scaffold assembled with an initially transcribed RNA sequence and a double-stranded DNA (dsDNA) sequence extending to up to 24 bps downstream of the TSS15–17. This experimental approach originates from the analysis of Pol II elongation complexes (ECs), preventing heterogenic sample conformations and making use of the tight DNA/RNA hybrid association with the polymerase[26,27]. The dsDNA scaffold was truncated on its downstream edge at a
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