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

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.