“…The observed CF-DNA interactions and footprinting experiments (Fig. 4i and 5a) are overall in good agreement with the results of Jackobel et al 35 who used DNA competitor oligonucleotides to map the minimal, sequence-specific CF binding region from bp −28 to bp −17.Fig. 5Biochemical characterization of CF promoter recognition. a ExoIII footprinting assay performed on the dsDNA transcription scaffold (−50 to +20) labelled either on the template or non-template strand.…”
RNA polymerase I (Pol I) assembles with core factor (CF) and Rrn3 on the rDNA core promoter for transcription initiation. Here, we report cryo-EM structures of closed, intermediate and open Pol I initiation complexes from 2.7 to 3.7 Å resolution to visualize Pol I promoter melting and to structurally and biochemically characterize the recognition mechanism of Pol I promoter DNA. In the closed complex, double-stranded DNA runs outside the DNA-binding cleft. Rotation of CF and upstream DNA with respect to Pol I and Rrn3 results in the spontaneous loading and opening of the promoter followed by cleft closure and positioning of the Pol I A49 tandem winged helix domain (tWH) onto DNA. Conformational rearrangement of A49 tWH leads to a clash with Rrn3 to initiate complex disassembly and promoter escape. Comprehensive insight into the Pol I transcription initiation cycle allows comparisons with promoter opening by Pol II and Pol III.
“…The observed CF-DNA interactions and footprinting experiments (Fig. 4i and 5a) are overall in good agreement with the results of Jackobel et al 35 who used DNA competitor oligonucleotides to map the minimal, sequence-specific CF binding region from bp −28 to bp −17.Fig. 5Biochemical characterization of CF promoter recognition. a ExoIII footprinting assay performed on the dsDNA transcription scaffold (−50 to +20) labelled either on the template or non-template strand.…”
RNA polymerase I (Pol I) assembles with core factor (CF) and Rrn3 on the rDNA core promoter for transcription initiation. Here, we report cryo-EM structures of closed, intermediate and open Pol I initiation complexes from 2.7 to 3.7 Å resolution to visualize Pol I promoter melting and to structurally and biochemically characterize the recognition mechanism of Pol I promoter DNA. In the closed complex, double-stranded DNA runs outside the DNA-binding cleft. Rotation of CF and upstream DNA with respect to Pol I and Rrn3 results in the spontaneous loading and opening of the promoter followed by cleft closure and positioning of the Pol I A49 tandem winged helix domain (tWH) onto DNA. Conformational rearrangement of A49 tWH leads to a clash with Rrn3 to initiate complex disassembly and promoter escape. Comprehensive insight into the Pol I transcription initiation cycle allows comparisons with promoter opening by Pol II and Pol III.
“…We further found that CX-5461 did not affect promoter occupancy by SL1 (TAF1B) or by UBF in NIH3T3 ( Supplementary Figures S2B and S3C ) or HEK293T cells ( Supplementary Figure S2C ). This excludes the possibility that the drug acts by preventing promoter recruitment of SL1 as was previously suggested ( 9 , 61 ).…”
Section: Resultsmentioning
confidence: 55%
“…Previous studies had suggested that the ability of CX-5461 to inhibit rDNA transcription was the result of inhibition of SL1 binding to the 47S promoter ( 9 , 61 ). However, our data clearly showed that neither SL1 nor UBF binding at the 47S or Spacer promoters was inhibited by CX-5461, excluding this as the mechanism of drug action.…”
In the search for drugs to effectively treat cancer, the last 10 years have seen a resurgence of interest in targeting ribosome biogenesis. CX-5461 is a potential inhibitor of ribosomal RNA synthesis that is now showing promise in phase I trials as a chemotherapeutic agent for a range of malignancies. Here, we show that CX-5461 irreversibly inhibits ribosomal RNA transcription by arresting RNA polymerase I (RPI/Pol1/PolR1) in a transcription initiation complex. CX-5461 does not achieve this by preventing formation of the pre-initiation complex nor does it affect the promoter recruitment of the SL1 TBP complex or the HMGB-box upstream binding factor (UBF/UBTF). CX-5461 also does not prevent the subsequent recruitment of the initiation-competent RPI–Rrn3 complex. Rather, CX-5461 blocks promoter release of RPI–Rrn3, which remains irreversibly locked in the pre-initiation complex even after extensive drug removal. Unexpectedly, this results in an unproductive mode of RPI recruitment that correlates with the onset of nucleolar stress, inhibition of DNA replication, genome-wide DNA damage and cellular senescence. Our data demonstrate that the cytotoxicity of CX-5461 is at least in part the result of an irreversible inhibition of RPI transcription initiation and hence are of direct relevance to the design of improved strategies of chemotherapy.
“…4). Sequence specificity is determined by proximal upstream bendability 15,65 and distal upstream recognition by UAF, which is linked to the PIC via CF and TBP. Divergent TFIIB reader-loop elements within Rrn7 are placed in the Pol I cleft, may play a role in duplex-destabilization and bind the melted template strand similar to observations in ITCs 17 .…”
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.
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