Biology subjects

Le, H. C.

Publications and source records attributed to Le, H. C..

4 recordsLinked to original sources

bioRxiv2026

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.

microbiology↗

bioRxiv2026

Coordinated synthesis of double-stranded DNA by a dual reverse transcriptase immune system

Defense-associated reverse transcriptase (DRT) systems mediate antiviral immunity through distinct modes of cDNA synthesis: Class 1 DRTs catalyze untemplated synthesis, whereas Class 2 DRTs polymerize noncoding RNA-templated products. However, how these distinct modes drive defense remains unclear. Here, we report that DRT3 immunity arises when Class 1 and Class 2 RT activities cooperate to produce self-complementary double-stranded DNA (dsDNA). DRT3a uses a 5'-ACACAC-3' RNA template to synthesize poly-(dTdG) repeats, whereas DRT3b synthesizes poly-(dCdA) repeats without any nucleic acid template. Cryo-electron microscopy reveals that DRT3b forms a hexamer and uses active-site-adjacent residues as deoxyadenosine and deoxycytidine gates to enforce alternating nucleotide addition, representing a unique example of amino acid-templated DNA polymerization. DRT3 is toxic in cells lacking RecBCD, implicating host recombination machinery in limiting dsDNA accumulation, and the phage-encoded RecBCD inhibitor Gam triggers DRT3-mediated abortive infection. These findings reveal how two polymerases with distinct templating strategies generate complementary DNA for defense.

microbiology↗

bioRxiv2023

Emergence of RNA-guided transcription factors via domestication of transposon-encoded TnpB nucleases

Transposon-encoded tnpB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination1-4. This widespread gene family was repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas125,6. We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRIS-PR-Cas. Here, using phylogenetics, structural predictions, comparative genomics, and functional assays, we uncover multiple instances of programmable transcription factors that we name TnpB-like nuclease-dead repressors (TldR). These proteins employ naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPRi technologies invented by humans7. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility8, phage susceptibility9, and host immunity10. Col-lectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of genes encoded by transposable elements, and reveals that RNA-guided transcription factors emerged long before the development of dCas9-based editors.

genetics↗

bioRxiv2023

Antagonistic conflict between transposon-encoded introns and guide RNAs

TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life, presumably due to the critical roles they play in transposon proliferation. IS605-family TnpB homologs function in bacteria as programmable homing endonucleases by exploiting transposon-encoded guide RNAs to cleave vacant genomic sites, thereby driving transposon maintenance through DSB-stimulated homologous recombination. Whether this pathway is conserved in other genetic contexts, and in association with other transposases, is unknown. Here we uncover molecular mechanisms of transposition and RNA-guided DNA cleavage by IS607-family elements that, remarkably, also encode catalytic, self-splicing group I introns. After reconstituting and systematically investigating each of these biochemical activities for a candidate IStron derived from Clostridium botulinum, we discovered sequence and structural features of the transposon-encoded RNA that satisfy molecular requirements of a group I intron and TnpB guide RNA, while still retaining the ability to be faithfully mobilized at the DNA level by the TnpA transposase. Strikingly, intron splicing was strongly repressed not only by TnpB, but also by the secondary structure of {omega}RNA alone, allowing the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts have evolved a sensitive equilibrium to balance competing and mutually exclusive activities that promote transposon maintenance while limiting adverse fitness costs on the host. Collectively, this work explains how diverse enzymatic activities emerged during the selfish spread of IS607-family elements and highlights molecular innovation in the multi-functional utility of transposon-encoded noncoding RNAs.

molecular biology↗