Structural and genetic dissection of RNA-guided gene repression by TnpB-derived transcription factors
- 1Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA. Howard Hughes Medical Institute, Columbia University, New York, NY,
Abstract
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.
Related
- Collaborative binding by diverse pioneer transcription factors amplifies Polycomb repression to preserve lineage fidelity
- tRNA-derived fragments restrict KSHV lytic replication through miRNA-like repression of host pro-viral signalling networks
- Haplotype-resolved genetic mapping using selfed progeny enables simultaneous genome assembly validation and trait dissection in a heterozygous plant
- Genetic dissection of a Solanum elaeagnifolium tertiary genepool introgression associated with 4-O-caffeoylquinic acid accumulation in eggplant
- Genetic dissection of soybean cyst nematode HG type 2.5.7 resistance reveals a multilocus architecture and favorable alleles from both parents
BibTeX
@misc{xiao2026structural,
title = {{Structural and genetic dissection of RNA-guided gene repression by TnpB-derived transcription factors}},
author = {R. Xiao and A. A. Casas-Ciniglio and H. C. Le and T. Wiegand and D. Xie and L. Chang and S. H. Sternberg},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.22.753285},
url = {https://www.biorxiv.org/content/10.64898/2026.09.22.753285v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Structural and genetic dissection of RNA-guided gene repression by TnpB-derived transcription factors
AU - R. Xiao
AU - A. A. Casas-Ciniglio
AU - H. C. Le
AU - T. Wiegand
AU - D. Xie
AU - L. Chang
AU - S. H. Sternberg
PY - 2026
DA - 2026/09/22
PB - bioRxiv
DO - 10.64898/2026.09.22.753285
UR - https://www.biorxiv.org/content/10.64898/2026.09.22.753285v1
AB - 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.
ER -