A stepwise 2′-hydroxyl activation mechanism for the bacterial transcription termination factor Rho helicase

The bacterial transcriptional termination factor Rho is a hexameric helicase that tracks along RNA and dissociates DNA-RNA hybrids. Here the activity of Rho is examined using nucleotide analog interference mapping, revealing that the helicase takes large, 7-nt steps, triggered by contacts with 2′OH in the tracked RNA substrate. The bacterial Rho factor is a ring-shaped ATP-dependent helicase that tracks along RNA transcripts and disrupts RNA-DNA duplexes and transcription complexes in its path. Using combinatorial nucleotide analog interference mapping (NAIM), we explore the topology and dynamics of functional Rho–RNA complexes and reveal the RNA-dependent stepping mechanism of Rho helicase. Periodic Gaussian distributions of NAIM signals show that Rho forms uneven productive interactions with the track nucleotides and disrupts RNA-DNA duplexes in a succession of large (∼7-nucleotide-long) discrete steps triggered by 2′-hydroxyl activation events. This periodic 2′-OH–dependent activation does not depend on the RNA-DNA pairing energy but is finely tuned by sequence-dependent interactions with the RNA track. These features explain the strict RNA specificity and contextual efficiency of the enzyme and provide a new paradigm for conditional tracking by a helicase ring.