Yeast Rrn7 and human TAF1B are TFIIB-related RNA polymerase I general transcription factors

Abstract

Eukaryotic and archaeal multisubunit RNA polymerases (Pols) are structurally related and require several similar components for transcription initiation. However, none of the Pol I factors were known to share homology with transcription factor IIB (TFIIB) or TFIIB-related proteins, key factors in the initiation mechanisms of the other Pols. Here we show that Rrn7, a subunit of the yeast Pol I core factor, and its human ortholog TAF1B are TFIIB-like factors. Although distantly related, Rrn7 shares many activities associated with TFIIB-like factors. Domain swaps between TFIIB-related factors show that Rrn7 is most closely related to the Pol III general factor Brf1. Our results point to the conservation of initiation mechanisms among multisubunit Pols and reveal a key function of yeast core factor/human SL1 in Pol I transcription.

Figure 1. Homology between S. cerevisiae Rrn7 and TFIIB family members

(A) HHpred search results. % Fold compares the predicted secondary structure of the template and query. E-value is the average expected number of non-homologous proteins with a score higher than the database match. (B) Schematic of TFIIB family organization. BHD, TFIIB homology domain; CTD, C-terminal domain; ZR, zinc ribbon; BR, B-reader; BL, B-linker. Arrows indicate the cyclin fold repeats, and the black squiggle indicates a TAF1B insertion. The number to the right indicates protein length. (C) Alignment of Rrn7, TAF1B, and TFIIB. Residues colored according to conservation. Blue highlight: TAF1B insertion.

Figure 2. Rrn7 ribbon and Pol I dock domain mutations affect Rrn7-Pol I interaction

(A) GST-Rrn7 fusion proteins. (B) Binding of Rrn7 Full length (FL), BHD, or CTD to immobilized Rpa135-Flag Pol I. (C) Binding of WT or Rrn7 ribbon mutant M1 to immobilized Pol I. (D) Sequence and secondary structure alignment of Rpa190 (Pol I), Rpb1 (Pol II), and Rpc1 (Pol III) dock domains. Residues are colored according to conservation and PSIpred predictions: helical (H, red), beta-sheet (E, blue), and coil (C, white). Known yeast Rpb1 secondary structure (1wtf_A) is shown. In the mutations Δ1 and Δ2, the deleted sequences were replaced by Gly-Ser-Gly. (E) Growth of yeast dock domain mutants. +++, WT growth; −, no growth. (F) Pol I with the indicated dock domain mutations was immobilized to a-Flag beads and tested for Rrn7 ribbon-binding. Panels B, C, F are Western blots probed with anti-Flag or -GST antibody.

Figure 3. Interchangeable TFIIB family domains

RRN7 yeast complementation assay with chimeric proteins containing (A) Rrn7 (grey) and TAF1B (red), (B) TFIIB (orange) or BRF1 (blue). (C) TFIIB and BRF1 yeast complementation assay with indicated Rrn7 domains (grey) in place of TFIIB or Brf1 domains. +++, WT growth; −, no growth.