(A) Bar diagrams of the Pol I regions involved in Rrn3 binding, with connecting lines as derived from the cryo-EM structure. α-helices 1 and 2 in the A14 TA domain are marked in dark red. Serine residues in the A43 cradle, A14 stretch and Rrn3 patch are shown above the corresponding bars, with Rrn3-S145 in red. (B) Close-up view of the Pol I–Rrn3 interaction in a similar orientation to that in Figure 6B. HEAT repeats in Rrn3 labelled H1 to H10. Dotted lines represent disordered regions in the Pol I and Rrn3 crystal structures. Boxed-text marks truncated regions in the yeast mutants of panel F. (C) Close-up view of the serine cradle in A43 that accommodates serine 145 in Rrn3. (D) Close-up view of A14 helix α2, which lies in the vicinity of Rrn3. (E) Pol I specific insertions in subunits A190 and A135 are shown in green. (F) Representative PICT images of the RFP-tagged anchor (upper row), GFP-tagged Rrn3 (middle row) and a zoom of a 2.6 × 2.6 µm square around anchoring platforms (bottom row) of different mutant strains. Below, quantification of the Rrn3-GFP recruitment score, normalized to the measurement of the wild-type strain (Mean ± SD, p-value * < 0.01 t-test). At the bottom, ChIP experiments showing the relative association of A190 (light) and Rrn3 (dark) to the rDNA promoter region. All ChIP experiments were normalized to the value of the wild-type strain in rich medium (Mean ± SD).