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Summary NusG/Spt5 proteins are the only transcription factors utilized by all cellular organisms. In enterobacteria, NusG antagonizes the transcription termination activity of Rho, a hexameric helicase, during the synthesis of ribosomal and actively translated mRNAs. Paradoxically, NusG helps Rho act on untranslated transcripts, including non-canonical antisense RNAs and those arising from translational stress; how NusG fulfills these disparate functions is unknown. Here, we demonstrate that NusG activates Rho by assisting helicase isomerization from an open-ring, RNA-loading state to a closed-ring, catalytically active translocase. A crystal structure of closed-ring Rho in complex with NusG reveals the physical basis for this activation and further explains how Rho is excluded from translationally competent RNAs. This study demonstrates how a universally conserved transcription factor acts to modulate the activity of a ring-shaped ATPase motor and establishes how the innate sequence bias of a termination factor can be modulated to silence pervasive, aberrant transcription. Graphical Abstract Highlights • NusG triggers an allosteric relay in Rho to promote activity on nonideal substrates • Rho can be reprogrammed to bypass NusG for transcription termination • NusE directly competes with Rho for binding to NusG to promote antitermination • NusG paralogs lack Rho-binding determinants, explaining functional specificity Lawson et al. show that NusG, a member of a universally conserved transcription factor family, helps isomerize the Rho transcription termination factor from an open-ring loading state to an active, closed-ring conformation. This action overrides the innate sequence bias of Rho to terminate aberrant transcriptional events emanating from translational stress. [ABSTRACT FROM AUTHOR]