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Sternberg Lab @sternberglab.bsky.social · 05/10/2026

2/2 We used sequencing and fluorescence reporter assays to uncover Arc’s role as a transcriptional repressor. It binds the transposon promoter and regulates gene expression, suggesting a new mechanism for transposon control!

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

13/13 — A joint effort between the Sternberg and Chang labs: Renjian Xiao, Americo Casas-Ciniglio, Henry Le, Tanner Wiegand, and Dan Xie, supervised by Leifu Chang and Sam Sternberg.

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

12/13 — In summary, TldR is a naturally evolved parallel to engineered CRISPRi. Repression of a single OppA variant shifts the relative contribution of remaining paralogs, which could influence which peptides a cell imports. What induces TldR natively remains unknown. www.biorxiv.org/content/10.6...

biorxiv.org

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 adapt...

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

11/13 — Indeed, mutating Q116 or L75 strongly impairs TldR-mediated transcription blocking in vitro, with a charge-introducing L75E largely abolishing it. Replacing the disordered Lid motif with GS linkers has no such effect.

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

10/13 — The 5′-TTTAA-3′ TAM is read out in a groove formed by the REC and WED domains, anchored by Q116 inserting into the major groove, with additional contributions from L75 and S74. Compared with ISDra2 TnpB, the recognition interface is reduced and reorganized.

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

9/13 — To see how TldR engages DNA, we determined a cryo-EM structure of the Efa₁TldR-gRNA-DNA ternary complex at 3.1 Å (99.7% identical to the V583 homolog). TldR is bilobed like TnpB, but its RuvC is degenerate and the Lid motif is disordered.

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

8/13 — Using AlphaFold 3, we modeled every V583 OppA paralog against both OppBCDF membrane complexes. Most paralogs dock confidently onto the TldR-associated transporter, while DdpA is matched to its own. Repressing one OppA could hand the shared transporter to a different partner.

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

7/13 — This holds well beyond Enterococcus: bacteria encode a median of 6 oppA paralogs per genome (IQR = 3-10), so V583 and its 10 loci are at the high end. Across the V583 paralogs the OppA fold is conserved, while the predicted substrate-binding pocket is not.

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

6/13 — Why would repressing one oppA activate others? E. faecalis V583 encodes a second oppABCDF operon plus eight standalone oppA homologs scattered across the genome, in diverse genetic contexts.

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

5/13 — Compared to a non-targeting gRNA control, oppA is the most significantly repressed gene in the transcriptome. Strikingly, a paralogous opp operon, an orphan oppA, and two other operons are up-regulated, with no off-target guide RNA matches to explain it.

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

4/13 — Deleting tldR and its gRNA produced virtually no genome-wide transcriptional changes, consistent with the modest native expression of TldR. Ectopic overexpression of TldR-gRNA from an agmatine-inducible plasmid, however, repressed oppA ~40-fold.

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

3/13 — The RNA-seq revised our picture of the locus. The oppA TSS sits upstream of the TldR target site, so the guide RNA target and its TAM fall inside the transcribed region rather than over the promoter, positioning TldR as a roadblock to elongating RNAP.

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

2/13 — oppF-associated TldRs (Wiegand et al. 2024) are predicted to target oppA, the substrate-binding protein of an oligopeptide ABC transporter, but this had not been tested in a native host. We therefore carried out total RNA-seq in E. faecalis V583.

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Sternberg Lab @sternberglab.bsky.social · 28/09/2026

1/13 — New pre-print from the Sternberg Lab in collaboration with Leifu Chang's Lab! TnpB nucleases have repeatedly given rise to TldRs, nuclease-dead RNA-guided transcription factors. Here we define the biological role of one TldR clade in its native host. Story: www.biorxiv.org/content/10.6...

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Sternberg Lab @sternberglab.bsky.social · 01/09/2026

Link to preprint post bsky.app/profile/ster...

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Sternberg Lab @sternberglab.bsky.social · 01/09/2026

Just published! IStrons are a unique class of transposable elements that pack three distinct functions in one nucleotide sequence. In this work, we used pooled library mutagenesis and high-throughput sequencing to systematically map the sequence and structural features that govern these functions.

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Reposted by Sternberg Lab

Nucleic Acids Research @narjournal.bsky.social · 28/08/2026

Congratulations to Edan Mortman and @sternberglab.bsky.social @shsternberg.bsky.social on this fascinating and important achievement! 🎉 Your work provides a compelling look at how selfish genetic elements balance their own propagation with the survival of their host. @columbiauniversity.bsky.social

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Reposted by Sternberg Lab

Jean-Michel Ané @jeanmichelane.bsky.social · 14/07/2026

Several faculty openings in cell and community synthetic biology at @uwmadison.bsky.social Some are at any level, so consider it for yourself and tell colleagues/stusdents. Great colleagues, state-of-the-art facilities, a great place to live and succeed in science! bact.wisc.edu/bacteriology...

bact.wisc.edu

Bacteriology is Hiring!

Bioeconomy Assistant Professor The Department of Bacteriology is seeking applicants at the Assistant Professor level to launch a research program in the New Bioeconomy. Applicants may be interested in...

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Sternberg Lab @sternberglab.bsky.social · 03/08/2026

Out now! In collaboration with Hiroshi Nishimasu's lab, we uncover how a dual reverse transcriptase immune system builds double-stranded DNA from both RNA and protein templates. www.cell.com/cell/abstrac... Previous thread for the preprint: bsky.app/profile/ster...

cell.com

Coordinated RNA- and protein-templated synthesis of double-stranded DNA by a dual reverse transcriptase immune system

Bacterial reverse transcriptases defend against viruses, yet how their DNA products stop infection remains unknown. Bacteriophage inhibition of a host recombination enzyme activates the DRT3 defense s...

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Sternberg Lab @sternberglab.bsky.social · 09/07/2026

11/11 Many thanks to all authors for their collective efforts in bringing this story to life! And a special thanks to @shsternberg.bsky.social & @piratefernandez.bsky.social for their mentorship and support throughout this project.

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Sternberg Lab @sternberglab.bsky.social · 09/07/2026

10/11 Our findings suggest a shared architectural logic underlying RNA-guided, tandem-repeat DNA synthesis across diverse bacterial Class 2 DRT systems. It also suggests that DRT10 is a structurally minimal version of the same strategy used by eukaryotic telomerase to achieve repeat addition.

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Sternberg Lab @sternberglab.bsky.social · 09/07/2026

9/11 And because the boundaries are geometric rather than sequence-specific, we could swap in templates from DRT3 and DRT9 and reprogram DRT10 to synthesize their signature repeats.

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Sternberg Lab @sternberglab.bsky.social · 09/07/2026

8/11 This anchored-boundary logic is structurally conserved across bacterial Class 2 DRT systems (DRT2, DRT3, DRT9), despite substantial divergence in protein sequence, ncRNA sequence, and oligomeric states.

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Sternberg Lab @sternberglab.bsky.social · 09/07/2026

7/11 How does DRT10 RT know where each repeat starts and ends? 2 RNA stem-loop anchors physically define the template boundaries. Geometry, not sequence, sets the template window, with microhomology on ncRNA sequence directing iterative template resetting between cycles of extension.

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Sternberg Lab @sternberglab.bsky.social · 09/07/2026

6/11 Symmetric structure, asymmetric function: both RT monomers are structurally identical and catalytically competent, but only one monomer produces the kilobase-length repeat DNA products, while the other generates short abortive products.

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Sternberg Lab @sternberglab.bsky.social · 09/07/2026

5/11 Two identical RT monomers bind opposite sides of a single, pseudo-symmetric, figure-eight-shaped ncRNA. The RNA is both the template and the scaffold holding the whole complex together.

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Sternberg Lab @sternberglab.bsky.social · 09/07/2026

4/11 How does DRT10 RT achieve such elegant tandem-repeat synthesis? We determined cryo-EM structures of two evolutionarily diverse DRT10 systems & found a surprise: the RT enzyme doesn't work alone...

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Sternberg Lab @sternberglab.bsky.social · 09/07/2026

3/11 Last year we reported that DRT10 RT repeatedly copies a short internal RNA template to generate long tandem-repeat DNA, which is conceptually similar to how telomerase extends chromosome ends. More details in our earlier preprint: www.biorxiv.org/content/10.1...

biorxiv.org

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 ...

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Sternberg Lab @sternberglab.bsky.social · 09/07/2026

1/11 New preprint from the Sternberg lab in collaboration with the Fernández lab! We are excited to share our structure-function study of DRT10, a bacterial defense-associated reverse transcriptase that synthesizes long tandem-repeat DNA.🧵 Read the full story here: www.biorxiv.org/content/10.6...

biorxiv.org

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

Defense-associated reverse transcriptases (DRTs) employ DNA synthesis to protect bacteria against phage infection[1][1],[2][2]. We previously showed that DRT10, a tripartite system comprising an RT, a...

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Sternberg Lab @sternberglab.bsky.social · 21/05/2026

8/8 Want the atomic details? The preprint has it all-- subcomplex structures, mutagenesis validations & a clear path toward engineering next-gen gene insertion tools. Huge congrats to co-first authors Giada and Seraina, and the whole Jinek & Sternberg lab teams! 🎉 tinyurl.com/typeI-CAST

tinyurl.com

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Sternberg Lab @sternberglab.bsky.social · 21/05/2026

7/8 The full mechanistic model 🏛️: every step gated, every checkpoint structural. A complete blueprint for engineering better gene insertion tools. 🛠️

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Sternberg Lab @sternberglab.bsky.social · 21/05/2026

6/8 But hooks alone aren't enough ⚡. TnsB's beta-barrel domain must physically dock onto TnsC and grab the target DNA — triggering an allosteric relay that orders disordered domains, remodels the catalytic site by ~11 Å, and switches TnsB on for DNA integration. No target = no chemistry.

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Sternberg Lab @sternberglab.bsky.social · 21/05/2026

5/8 This "super-ring" clamps the targeting complex and fixes the integration site 49 nt away. The transposase (TnsAB) arrives next, dangling by tiny C-terminal "hooks" 🪝 off the TnsC ring.

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Sternberg Lab @sternberglab.bsky.social · 21/05/2026

4/8 The machine operates through a cascade of fidelity checkpoints 🔒🔒🔒. First the Cascade-TniQ complex forms a complete R-loop. The Cas8 subunit rotates ~90° to lock onto the target DNA, only then does TnsC assemble into a heptameric ring 💍 around the downstream DNA, contacting both TniQ and Cas8.

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Sternberg Lab @sternberglab.bsky.social · 21/05/2026

3/8 We solved the structure of a 1.2 MDa molecular machine caught in the act of inserting DNA into a genome. Here's what we found. 🧵

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Sternberg Lab @sternberglab.bsky.social · 21/05/2026

2/8 The type I-F PseCAST system is the most active in human cells to date, but how it achieves such precision was unknown. Until now. tinyurl.com/typeI-CAST 👀

tinyurl.com

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Sternberg Lab @sternberglab.bsky.social · 21/05/2026

1/8 🚨 New preprint from the @sternberglab.bsky.social & @martinjinek.bsky.social labs! CRISPR-associated transposases (CASTs) insert large DNA cargoes at precise genomic locations — no double-strand breaks needed.

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Sternberg Lab @sternberglab.bsky.social · 12/05/2026

Be sure to also check out the elegant concurrent work from Alex Gao's group: science.org/doi/10.1126/...

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Sternberg Lab @sternberglab.bsky.social · 12/05/2026

9/9 This was a huge team effort! Big thanks to Kanta, Junichiro, and the Nishimasu lab for adjusting their meeting schedules to collaborate across time zones. Equal gratitude goes out to the Sternberg lab for the discussions and shared curiosity that shaped this work!

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Sternberg Lab @sternberglab.bsky.social · 12/05/2026

8/9 How does this drive defense? Phage λ gam triggers DRT3 cell death by inhibiting RecBCD, which normally degrades the dsDNA product to keep toxicity in check. A Tn-seq screen also pulled out greA, hinting at a transcription-related arm of immunity.

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Sternberg Lab @sternberglab.bsky.social · 12/05/2026

7/9 Together, DRT3a and DRT3b make self-complementary strands that anneal into dsDNA, confirmed directly by a PicoGreen assay showing time-dependent dsDNA accumulation only when both enzymes are catalytically active.

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Sternberg Lab @sternberglab.bsky.social · 12/05/2026

6/9 E22 acts as a gatekeeper that excludes dTTP and dGTP via electrostatic repulsion. R241 acts as a specificity switch: once an A is at the -1 position, R241 swings over and only allows a C next. DRT3b functions via amino acid-templated DNA polymerization!

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Sternberg Lab @sternberglab.bsky.social · 12/05/2026

5/9 To understand how, the Nishimasu Lab solved a cryo-EM structure of DRT3b. It assembles into a hexamer with bound poly-(dCdA) DNA. Two residues near the active-site enforce the alternating sequence.

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Sternberg Lab @sternberglab.bsky.social · 12/05/2026

4/9 DRT3b’s synthesis activity is novel for this family of bacterial reverse transcriptases. It synthesizes precise poly-(dCdA) repeats with no nucleic acid template at all. The enzyme alone, with only dCTP and dATP, generates dinucleotide repeats.

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Sternberg Lab @sternberglab.bsky.social · 12/05/2026

3/9 DRT3a uses a conserved 5'-ACACAC-3' template in the ncRNA to synthesize poly-(dTdG), similar to our previous work with DRT9 and DRT10. Mutating this motif abolishes cDNA production and phage defense.

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Sternberg Lab @sternberglab.bsky.social · 12/05/2026

2/9 Using cDIP-seq, we discovered that cells expressing DRT3 accumulate two distinct cDNA species: poly-(dTdG) and poly-(dCdA) dinucleotide repeats. Biochemical sequencing nicely assigns each product to one enzyme. DRT3a makes poly-(dTdG), DRT3b makes poly-(dCdA).

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Sternberg Lab @sternberglab.bsky.social · 12/05/2026

1/9 New preprint from the Sternberg Lab in collaboration with the Nishimasu Lab! We uncover how the DRT3 antiphage immune system pairs two reverse transcriptases, one RNA-templated and one protein-templated, to build a double-stranded DNA effector. doi.org/10.64898/202...

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