Biology subjects

Sternberg, S. H.

Publications and source records attributed to Sternberg, S. H..

At least 19 recordsLinked to original sources

bioRxiv2026

Arc represses gene expression in IS605-family transposons

Bacterial insertion sequences (IS) are compact transposable elements that encode proteins required for their mobility and maintenance, yet many also encode accessory proteins with poorly understood functions. For example, IS605-family elements often encode a transposase called TnpA and an RNA-guided nuclease called TnpB that supports transposon maintenance, alongside an additional ribbon-helix-helix protein named Arc. Though the roles of TnpA and TnpB have been extensively studied in recent years, the enigmatic function of Arc has not been investigated. Here, we show that Arc acts as a transcriptional repressor to directly bind the transposon's native promoter sequence regulating TnpA and TnpB gene expression. By systematically testing Arc-containing IS605 elements, we identified a conserved binding pattern at intergenic transposon sequences neighboring protein-coding genes through chromatin immunoprecipitation and sequencing analyses. We then used fluorescence reporter assays and demonstrated that these intergenic sequences function as strong promoters, and that the presence of Arc dramatically reduces their gene expression. Together, these findings identify Arc as a transposon-encoded transcriptional repressor, revealing a regulatory layer that may promote long-term persistence of IS605-family elements by keeping their activity in check. The widespread association of Arc homologs with diverse mobile elements and cellular genes suggests that these compact regulators may more broadly restrain the expression of neighboring genetic machinery across varied genomic contexts. neighboring genetic machinery across varied genomic contexts.

molecular biology↗

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

Mechanism of tandem-repeat DNA synthesis by an antiviral reverse transcriptase

Defense-associated reverse transcriptases (DRTs) employ DNA synthesis to protect bacteria against phage infection1,2. We previously showed that DRT10, a tripartite system comprising an RT, a noncoding RNA (ncRNA), and a SLATT effector protein, catalyzes protein-primed, tandem-repeat DNA synthesis in a mechanism strikingly analogous to eukaryotic telomerase3. However, the structural basis by which the RT-ncRNA complex directs repeat addition processivity and controls repeat length remains unknown. Here we present cryo-EM reconstructions of two evolutionarily diverse DRT10 RT-ncRNA systems that reveal an unanticipated 2:1 architecture, wherein two RT monomers bind opposite sides of a single, pseudo-symmetric ncRNA. Biochemical experiments demonstrate that each RT monomer reverse transcribes the template encoded on its respective side of the ncRNA, but only one generates the long repetitive product, with the template sequence defined by the distance between two flanking stem-loop anchors. Together with earlier studies of DRT2, DRT3, and DRT94-6, our findings identify a conserved mechanistic logic underlying ncRNA-templated tandem-repeat synthesis across Class 2 UG antiviral systems, despite vastly different architectural solutions.

microbiology↗

bioRxiv2026

Structural basis of RNA-guided DNA integration by type I CRISPR-associated transposases

CRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like transpososome complex1,2. Understanding the detailed mechanisms of this elaborate process is paramount to engineering CAST systems into programmable genetic tools3-6. The type I-F Pseudoalteromonas CAST (PseCAST) displays the highest activity in mammalian cells to date7 and has been the subject of extensive directed evolution8, but efforts to rationally engineer further improvements have been hampered by critical gaps in our understanding of transpososome assembly and activation9. Here we use cryo-EM structural analysis, validated by DNA transposition assays, to visualize the PseCAST system in a series of functional states that define the stepwise mechanism of RNA-guided DNA integration. The structure of a target DNA-bound Cascade-TniQ-TnsC complex reveals that conformational changes induced by R-loop formation are coupled to target DNA stabilization and TnsC heptamerization, which in turn recruits the TnsAB transposase via conserved interactions with its C-terminal tail. Finally, the structure of the 1.2 MDa PseCAST transpososome holocomplex reveals specific TnsC-TnsB and TnsB-target DNA interactions that drive allosteric remodelling of the TnsB catalytic site to activate donor DNA integration. Together, these findings establish a unified structural and mechanistic blueprint for RNA-guided DNA integration and lay the foundation for engineering next-generation DNA insertion systems for genome editing applications.

molecular biology↗

bioRxiv2026

Transposon end recognition and excision mechanisms of type I-F CRISPR-associated transposases

CRISPR-associated transposons (CASTs) are Tn7-like elements that have co-opted RNA-guided CRISPR effectors for targeted DNA insertion. CASTs have been adapted as genome editing tools for programmable, site-specific integration. Among them, the type I-F system from Pseudoalteromonas (PseCAST) shows uniquely robust activity in human cells, yet its mechanistic basis remains poorly understood. Here, we present structural and biochemical analysis of the PseCAST transposase TnsAB. Biochemical reconstitution of transposon DNA excision defines key characteristics of the transposition mechanism. Cryogenic electron microscopy (cryo-EM) structures of PseTnsAB paired-end complexes reveal molecular determinants of transpososome assembly, transposon end recognition and cleavage. We validate these findings using biochemical and in vivo assays of structure-based transposase mutants, and provide mechanistic insights into the enhanced activity of a laboratory-evolved TnsAB variant. Together, our studies highlight molecular features underlying the efficiency of natural and engineered type I-F transposases and establish a mechanistic framework for their continued rational optimization.

biochemistry↗

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↗

bioRxiv2026

Large-scale mutational analysis uncovers molecular mechanisms governing dual RNA functions in transposons

Transposons are among the most abundant mobile genetic elements in nature. IStrons are a unique class of transposons, encoding a transposase for transposon mobility, an RNA-guided nuclease for maintenance, and a self-splicing group I intron for element removal from host mRNA. However, it is unclear how a single polynucleotide sequence balances these distinct biochemical functions. Here we employed pooled library mutagenesis coupled with high-throughput sequencing to systematically dissect the molecular determinants of IStron transposition, RNA-guided DNA cleavage, and self-splicing. We found that the terminal trinucleotide of the transposon right end is constrained by all three functions, identifying a molecular convergence point. Cross-assay comparisons revealed that most variants maintained or lost activity across multiple assays simultaneously. However, a subset selectively retained one activity while losing another, revealing an antagonism between DNA cleavage and splicing governed by guide RNA structural stability. Increased GC content at the base of the guide RNA 3 terminal stem-loop abolished splicing while maintaining DNA cleavage, and the stably folded guide RNA sterically occludes alternative splice sites, ensuring splicing accuracy across variable flanking contexts. Thus, IStron transcripts face an inherent trade-off between guide RNA maturation and splicing, with RNA structural stability as the primary determinant of pathway choice.

molecular biology↗

bioRxiv2025

Antiviral reverse transcriptases reveal the evolutionary origin of telomerase

Defense-associated reverse transcriptases (DRTs) employ diverse and distinctive mechanisms of cDNA synthesis to protect bacteria against viral infection. However, much of DRT family diversity remains unstudied. Here we identify a new antiviral defense system, DRT10, that associates with a non-coding RNA (ncRNA) to catalyze processive, protein-primed synthesis of tandem-repeat DNA. Repeat addition is dictated by sequence and structural features of the ncRNA that have direct parallels in the RNA component of telomerase. Remarkably, a phylogenetic analysis of RTs across domains of life reveals an unexpected link between DRT10 and telomerase that is further supported by structural comparisons and mechanistic evidence. These findings expand the repertoire of reverse transcription mechanisms in antiviral defense and point to a bacterial origin for telomerase. One-Sentence SummaryInsights from antiviral defense systems reveal an unexpected bacterial origin for the mechanism of chromosome maintenance by eukaryotic telomerase.

molecular biology↗

bioRxiv2025

Temperate phages enhance host fitness via RNA-guided flagellar remodeling

Bacterial flagella drive motility and chemotaxis while also playing critical roles in host-pathogen interactions, as their oligomeric subunit, flagellin, is specifically recognized by the mammalian immune system and flagellotropic bacteriophages. We recently discovered a family of phage-encoded, RNA-guided transcription factors known as TldR that regulate flagellin expression. However, the biological significance for this regulation, particularly in the context of host fitness, remained unknown. By focusing on a human clinical Enterobacter isolate that encodes a Flagellin Remodeling prophage (FR{varphi}), here we show that FR{varphi} exploits the combined action of TldR and its flagellin isoform to dramatically alter the flagellar composition and phenotypic properties of its host. This transformation has striking biological consequences, enhancing bacterial motility and mammalian immune evasion, and structural studies by cryo-EM of host- and prophage-encoded filaments reveal distinct architectures underlying these physiological changes. Moreover, we find that FR{varphi} improves colonization in the murine gut, illustrating the beneficial effect of prophage-mediated flagellar remodeling in a host-associated environment. Remarkably, flagellin-regulating TldR homologs emerged multiple times independently, further highlighting the strong selective pressures that drove evolution of RNA-guided flagellin control. Collectively, our results reveal how RNA-guided transcription factors emerged in a parallel evolutionary path to CRISPR-Cas and were co-opted by phages to remodel the flagellar apparatus and enhance host fitness.

microbiology↗

bioRxiv2025

Exapted CRISPR-Cas12f homologs drive RNA-guided transcription

Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma ({sigma}) factors, leading to functional DNA engagement by RNA polymerase (RNAP)1. Although the seven {sigma} factors in E. coli have been extensively characterized2, Bacteroidetes species encode dozens of specialized, extracytoplasmic function {sigma} factors ({sigma}E) whose precise roles are unknown, pointing to additional layers of regulatory potential3. Here we uncover an unprecedented mechanism of RNA-guided gene activation involving the coordinated action of {sigma}E factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically-linked dCas12f and {sigma}E homologs in E. coli using RIP-seq and ChIP-seq experiments, revealing systems that exhibited robust guide RNA enrichment and DNA target binding with a minimal 5'-G target-adjacent motif (TAM). Recruitment of {sigma}E was dependent on dCas12f and guide RNA (gRNA), suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-{sigma}E ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-{sigma}E complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription (RGT) that embodies natural features reminiscent of CRISPRa technology developed by humans4,5.

molecular biology↗

bioRxiv2025

Structural basis of RNA-guided transcription by a dCas12f-σE-RNAP complex

RNA-guided proteins have emerged as critical transcriptional regulators in both natural and engineered biological systems by modulating RNA polymerase (RNAP) and its associated factors1-3. In bacteria, di-verse clades of repurposed TnpB and CRISPR-associated proteins repress gene expression by blocking transcription initiation or elongation, enabling non-canonical modes of regulatory control and adaptive immunity1,4,5. Intriguingly, a distinct class of nuclease-dead Cas12f homologs (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors ({sigma}E)6, though the molecular basis has remained elusive. Here we reveal a novel mode of RNA-guided transcription initiation by determining cryo-electron microscopy structures of the dCas12f-{sigma}E system from Flagellimonas taeanensis. We captured multiple conformational and compositional states, including the DNA-bound dCas12f-{sigma}E-RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to {sigma}E-RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of {sigma}E-dependent promoter recognition, these studies show that recognition of the -35 element is largely supplanted by CRISPR-Cas targeting, while the melted -10 element is stabilized through unusual stacking interactions rather than insertion into the typical recognition pocket. Collectively, this work provides high-resolution insights into an unexpected mechanism of RNA-guided transcription, expanding our understanding of bacterial gene regulation and opening new avenues for pro-grammable transcriptional control.

biochemistry↗

bioRxiv2025

Structural basis for Lamassu-based antiviral immunity and its evolution from DNA repair machinery

Bacterial immune systems exhibit remarkable diversity and modularity, as a consequence of the continuous selective pressures imposed by phage predation. Despite recent mechanistic advances, the evolutionary origins of many antiphage immune systems remain elusive, especially for those that encode homologs of the Structural Maintenance of Chromosomes (SMC) superfamily, which are essential for chromosome maintenance and DNA repair across domains of life. Here, we elucidate the structural basis and evolutionary emergence of Lamassu, a bacterial immune system family featuring diverse effectors but a core conserved SMC-like sensor. Using cryo-EM, we determined structures of the Vibrio cholerae Lamassu complex in both apo- and dsDNA-bound states, revealing unexpected stoichiometry and topological architectures. We further demonstrate how Lamassu specifically senses dsDNA in vitro and phage replication origins in vivo, thereby triggering the formation of LmuA tetramers that activate the Cap4 nuclease domain. Our findings reveal that Lamassu evolved via exaptation of the bacterial Rad50-Mre11 DNA repair system to form a compact, modular sensor for viral replication, exemplifying how cellular machinery can be co-opted for novel immune functions.

microbiology↗

bioRxiv2025

Protein-primed DNA homopolymer synthesis by an antiviral reverse transcriptase

Bacteria defend themselves from viral predation using diverse immune systems, many of which sense and target foreign DNA for degradation1. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by leveraging DNA synthesis instead2,3. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene, leading to expression of an antiviral effector protein, Neo4,5. It remains unknown whether similar mechanisms of defense are employed by other DRT families. Focusing on DRT9, here we uncover an unprecedented mechanism of DNA homopolymer synthesis, in which viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell to drive abortive infection and population-level immunity. Cryo-EM structures reveal how a conserved noncoding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and RNA-templated poly-dA synthesis. Remarkably, biochemical and functional experiments identify conserved tyrosine residues within the reverse transcriptase itself that prime DNA synthesis, leading to the formation of high-molecular weight protein-DNA covalent adducts. Synthesis of poly-dA in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers of DRT9. Collectively, our work unveils a novel nucleic acid-driven defense system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.

molecular biology↗

bioRxiv2025

Relaxed DNA substrate specificity of transposases involved in programmed genome rearrangement

During post-zygotic development, the ciliate Oxytricha trifallax undergoes massive programmed genome rearrangement that involves over 225,000 DNA cleavage and joining events. An Oxytricha family of Tc1/mariner transposons, known as Telomere-Bearing Elements (TBEs), encodes a transposase that has been implicated in rearrangement, but its high copy number (>34,000 paralogs) has precluded genetic strategies to investigate its DNA recognition properties directly in Oxytricha. Here, we developed a heterologous strategy to assay TBE transposase expression and activity in E. coli, revealing highly promiscuous DNA cleavage properties. Systematic ChIP-seq experiments allowed us to define the DNA binding specificities of multiple distinct transposase subfamilies, which exhibited a binding and cleavage preference for short, degenerate sequence motifs that resemble features present within the TBE transposon ends. The relaxed sequence preference is striking for autonomous transposases, which typically recognize their end sequences with strict specificity to avoid compromising host fitness. Finally, we developed a custom antibody to investigate TBE transposases in their native environment and found that they precisely localize to the developing nucleus exclusively during the rearrangement process. Collectively, this work establishes a robust heterologous workflow for the biochemical investigation of enzymes that have been repurposed for large-scale genome rearrangements.

molecular biology↗

bioRxiv2024

Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells

Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in a heterogeneous mixture of undesirable editing outcomes. We recently leveraged a type I-F CRISPR-associated transposase (CAST) from the Pseudoalteromonas Tn7016 transposon (PseCAST) for DSB-free, RNA-guided DNA integration in human cells, taking advantage of its programmability and large payload capacity. PseCAST is the only characterized CAST system that has achieved human genomic DNA insertions, but multiple lines of evidence suggest that DNA binding may be a critical bottleneck that limits high-efficiency activity. Here we report structural determinants of target DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), which revealed novel subtype-specific interactions and RNA-DNA heteroduplex features. By combining our structural data with target DNA library screens and rationally engineered protein mutations, we uncovered CAST variants that exhibit increased integration efficiency and modified PAM stringency. Structure predictions of key interfaces in the transpososome holoenzyme also revealed opportunities for the design of hybrid CASTs, which we leveraged to build chimeric systems that combine high-activity DNA binding and DNA integration modules. Collectively, our work provides unique structural insights into type I-F CAST systems while showcasing multiple diverse strategies to investigate and engineer new RNA-guided transposase architectures for human genome editing applications.

molecular biology↗

bioRxiv2024

Evolutionary origins of archaeal and eukaryotic RNA-guided RNA modification in bacterial IS110 transposons

Transposase genes are ubiquitous in all domains of life and provide a rich reservoir for the evolution of novel protein functions. Here we report deep evolutionary links between bacterial IS110 transposases, which catalyze RNA-guided DNA recombination using bridge RNAs, and archaeal/eukaryotic Nop5-family proteins, which promote RNA-guided RNA 2-O-methylation using C/D-box snoRNAs. Based on conservation in the protein primary sequence, domain architecture, and three-dimensional structure, as well as common architectural features of the non-coding RNA components, we propose that programmable RNA modification emerged via exaptation of components derived from IS110-like transposons. Alongside recent studies highlighting the origins of CRISPR-Cas9 and Cas12 in IS605-family transposons, these findings underscore how recurrent domestication events of transposable elements gave rise to complex RNA-guided biological mechanisms.

evolutionary biology↗

bioRxiv2024

De novo gene synthesis by an antiviral reverse transcriptase

Bacteria defend themselves from viral infection using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling-circle reverse transcription of a non-coding RNA (ncRNA). Unbiased profiling of RT-associated RNA and DNA ligands in DRT2-expressing cells revealed that reverse transcription generates concatenated cDNA repeats through programmed template jumping on the ncRNA. The presence of phage then triggers second-strand cDNA synthesis, leading to the production of long double-stranded DNA. Remarkably, this DNA product is efficiently transcribed, generating messenger RNAs that encode a stop codon-less, never-ending ORF (neo) whose translation causes potent growth arrest. Phylogenetic analyses and screening of diverse DRT2 homologs further revealed broad conservation of rolling-circle reverse transcription and Neo protein function. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA. One-Sentence SummaryBacterial reverse transcriptases synthesize extrachromosomal genes via rolling-circle amplification to confer potent antiviral immunity.

molecular biology↗

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↗