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 ( approximately 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.