Structural and genetic dissection of RNA-guided gene repression by TnpB-derived transcription factors

TnpB nucleases, the evolutionary progenitors of CRISPR-associated Cas12 enzymes, are transposon-encoded, RNA-guided endonucleases found throughout bacteria. Independent of the trajectory towards adaptive immunity, TnpB nucleases have also recurrently given rise to TnpB-like nuclease-dead repressors (TldRs), a family of programmable RNA-guided transcription factors, though the physiological roles of most TldR clades remain unclear. Recently, we identified a TldR clade associated with bacterial ABC transporter operons, but this clade has not been characterized in any native host, leaving the biological significance of its predicted regulatory function unknown. Here, we demonstrate direct in vivo repression of the oligopeptide permease ( opp ) binding protein OppA in Enterococcus faecalis , implicating TldR in shaping the substrate repertoire of bacterial peptide import machinery. Phylogenetic and comparative genomic analyses reveal that bacterial genomes typically encode multiple, structurally conserved but functionally diversified OppA paralogs, suggesting that TldR-mediated repression could enable selective tuning of transporter composition. To define the structural basis of this regulatory specificity, we used cryo-electron microscopy to capture an oppF -associated TldR in multiple functional states, revealing a conserved bilobed architecture and a TAM recognition mechanism inherited from TnpB ancestors. Together, these findings define the structural, genetic, and evolutionary basis of a widespread RNA-guided regulatory system domesticated from mobile genetic elements to tune peptide transport in bacteria.