Efficient termination of transcription by RNA polymerase I requires a conserved hairpin of the ribosomal RNA precursor

Abstract

RNA polymerase I (Pol I) synthesizes ribosomal RNA precursor (pre-rRNA), which comprises most of RNA in eukaryotic cells. Despite decades of investigation, there is still no consensus on what causes Pol I transcription termination. Here, we show that efficient termination by Pol I, paused by termination roadblock protein, is caused by RNA hairpin of the nascent pre-rRNA. Hairpin-dependent termination takes place at a physiological rate and does not require trans-acting factors. The function of the roadblock protein and the T-rich sequence is to synergistically cause deep backtracking of Pol I toward the termination RNA hairpin. Simultaneously, Pol I is catalytically inactivated, preventing rescue from backtracking through RNA cleavage and thus committing Pol I to termination. Termination RNA hairpins are present in most of Pol I terminators of eukaryotes, suggesting conservation of the RNA hairpin-dependent mechanism of Pol I transcription termination. We propose a simple model that unifies previous findings.

Fig. 1.. Conserved RNA hairpin of the pre-rRNA causes termination of stopped EC.

(A) Scheme of the end of pre-rRNA gene, where Pol I terminates transcription after being stopped by Reb1 or Nsi1 roadblock. Blue arrows show the part of native hairpin used in the assembled ECs. Details of all ECs are shown in fig. S1B. (B) Scheme of the experiment for analysis of Pol I termination. Note that RNA13 in EC13 is radiolabeled at the 3′ end using the natural intrinsic cleavage activity of Pol I. (C) Reb1- or Nsi1-stopped Pol I does not terminate on the T-rich sequence. The scheme of the EC is shown next to the gel (EC^a in fig. S1B). Hereinafter, radioactive GMPs of the RNA (purple) are in bold, and quantitation of the RNA release is below the gels [super/(super + beads); means ± SD from at least three independent experiments]. (D) In the presence of the conserved pre-rRNA hairpin (termination hairpin), Reb1- or Nsi1-stopped EC efficiently and rapidly terminates transcription. The scheme of the EC is shown next to the gel (EC^c in fig. S1B). Blue arrows show the part of the termination hairpin coded in this EC [see (A)]. (E) RNA hairpin–dependent termination takes place at physiological rate. ECs were stopped by either Reb1, Nsi1, or AP at the position of the natural stop by Reb1/Nsi1, in the presence or absence of the termination hairpin (EC^a,b,c,d in fig. S1B). Note that the fastest time point possible in this experiment is 20 s. Data points are the mean of at least three independent experiments; error bars are ±SD. The raw data are presented in table S1.

Fig. 2.. Reb1-stopped EC undergoes deep backtracking along the T-rich sequence with inactivation of RNA cleavage activity.

(A) Exo III footprinting of the rear edge of the Reb1-stopped EC (ECⁿ in fig. S1B). The template DNA is radiolabeled at the 5′ end. The 3′ end of the non–template DNA contains phosphorothioate bonds to prevent Exo III digestion. The observed undersaturation of the Reb1-DNA complexes with the EC is expected from the way of EC assembly (see Materials and Methods). nt, nucleotides. (B and C) Stopping the EC by AP in the template DNA does not cause quick termination (see also Fig. 1E), which is restored by Reb1, but only when it is bound to its binding site (EC^d,e in fig. S1B). Note that Pol I misincorporates at the AP site, whereas addition of Reb1 abolishes this misincorporation, indicating correct binding on the AP template. (D) The roadblock and T-rich sequence are only required to efficiently bring the EC in the proximity of the termination RNA hairpin because rapid termination is achieved when the EC is stopped 8 bp downstream of the RNA hairpin by either AP or Reb1 (EC^f,g in fig. S1B). (E) Mutagenesis of the T-rich sequence inhibits termination, likely by affecting backtracking of the roadblocked EC toward the termination RNA hairpin (EC^l,m in fig. S1B) and strengthening of the RNA/DNA hybrid of terminating EC (EC^k in fig. S1B). The control with the native T-rich sequence is shown in fig. S2D. (F) Efficient cleavage activity of Pol I is inhibited during deep backtracking. ECs were one-step walked in subsets of NTPs to the positions indicated or stopped by Reb1 roadblock and washed, and cleavage was initiated by addition of Mg²⁺. Note that initial RNA13 was labeled at the 3′ end before walking (bold Gs in the scheme above; EC^a in fig. S1B).

Fig. 3.. Model of termination by Pol I.

Kinetics and functional discrimination between transcribing and terminating Pol I ECs. Note that the change in Rpa12 (blue) position is hypothetical and only shown to reflect inactivation of RNA cleavage activity. We suggest that RNA hairpin–dependent termination is the main termination pathway, which is further supported by fail-safe “torpedo” and release factor PTRF termination mechanisms when Pol I traffic jams may obstruct the RNA hairpin–dependent pathway (see Discussion).