Sternberg Lab

1/7 Last week the Sternberg Lab welcomed ~40 junior & senior high school AP Research students from the East Meadow school district (and their incredible teachers) into our lab at Columbia University.

6/7 And they enjoyed this agarose gel meme, made especially for our young friends ;)

7/7 As a lab we believe in sharing both our science and our research knowledge with others, and hope we’ve inspired a few minds! Looking forward to the next visit!

1/7 Last week the Sternberg Lab welcomed ~40 junior & senior high school AP Research students from the East Meadow school district (and their incredible teachers) into our lab at Columbia University.

4/7 Students also had the opportunity to check out hands-on work in the lab with stations on robotics, microbiology, cryoEM, phages, gel electrophoresis, HT sequencing, and human tissue culture.

5/7 Students also got to take home some pretty cool 3D printed bacteriophages!

1/7 Last week the Sternberg Lab welcomed ~40 junior & senior high school AP Research students from the East Meadow school district (and their incredible teachers) into our lab at Columbia University.

2/7 We shared our personal research backgrounds with the students, and they had the opportunity to present elevator pitches on their current research projects.

3/7 The East Meadow students are asking big questions: How does TikTok shape teen identity? Can AI be trusted for health info? How do climate shortages affect NYC restaurants? We had the chance to discuss these topics (and more) and give feedback on the research process.

nature.comAntiviral immunity via telomerase-like reverse transcription in bacteria - NatureNature - Antiviral immunity via telomerase-like reverse transcription in bacteria

1/5 Telomerase has long been linked to chromosome maintenance in eukaryotes. Now, we show that telomerase-like activity is also present in bacteria, shedding new light on its ancient origins. Excited to share that our work on DRT10 is now out in @nature.com! www.nature.com/articles/s41...

biorxiv.orgMechanism of tandem-repeat DNA synthesis by an antiviral reverse transcriptaseDefense-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...

4/5 DRT10 biochemistry and structure (with @piratefernandez.bsky.social): bsky.app/profile/ster...

Sternberg Lab@sternberglab.bsky.social

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...

5/5 Huge congratulations to all the authors for their incredible work bringing this story together! It was a fantastic collaborative effort spanning time zones and continents.

nature.comAntiviral immunity via telomerase-like reverse transcription in bacteria - NatureNature - Antiviral immunity via telomerase-like reverse transcription in bacteria

2/5 This work combines two complementary stories: one uncovering DRT10 biology and its link to telomerase, and the other exploring its structure and biochemical mechanism. We're excited to bring these findings together into a comprehensive picture of DRT10 function and evolution!

biorxiv.orgAntiviral reverse transcriptases reveal the evolutionary origin of telomeraseDefense-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 ...

3/5 For a deeper dive into the science, check out our original preprint threads below: DRT10 biology and evolution (with @pentamorfico.bsky.social and @rafomics.bsky.social): bsky.app/profile/ster...

Sternberg Lab@sternberglab.bsky.social

1/10 Genome maintenance by telomerase is a fundamental process in nearly all eukaryotes. But where does it come from? Today, we report the discovery of telomerase homologs in a family of antiviral RTs, revealing an unexpected evolutionary origin in bacteria. www.biorxiv.org/content/10.1...

biorxiv.orgArc represses gene expression in IS605-family transposonsBacterial 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 fu...

1/2 Here we report the activity of Arc, a 50-aa DNA-binding protein encoded within IS605-family transposons. www.biorxiv.org/content/10.6...

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!

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...

biorxiv.orgStructural and genetic dissection of RNA-guided gene repression by TnpB-derived transcription factorsTnpB nucleases, the evolutionary progenitors of CRISPR-associated Cas12 enzymes, are transposon-encoded, RNA-guided endonucleases found throughout bacteria. Independent of the trajectory towards adapt...

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...

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

1 Sept

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.

academic.oup.comLarge-scale mutational analysis uncovers molecular mechanisms governing dual RNA functions in transposonsAbstract. Transposons are among the most abundant mobile genetic elements in nature. IStrons are a unique class of transposons, encoding a transposase for

1 Sept

Link to paper! academic.oup.com/nar/article/...

@jeanmichelane.bsky.social

1 Sept

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

Sternberg Lab@sternberglab.bsky.social

New preprint from the Sternberg lab! We used pooled oligonucleotide library mutagenesis + high-throughput sequencing to systematically dissect the molecular determinants of IStron transposition, RNA-guided DNA cleavage, and self-splicing. www.biorxiv.org/content/10.6...

Sternberg Lab reposted

Nucleic Acids Research

@narjournal.bsky.social

28 Aug

Replying to @narjournal.bsky.social

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

Sternberg Lab reposted

Jean-Michel Ané

14 July

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.eduBacteriology 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...

cell.comCoordinated RNA- and protein-templated synthesis of double-stranded DNA by a dual reverse transcriptase immune systemBacterial 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...

3 Aug

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...

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.

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.

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.

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.

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.

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.

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...

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.

2/11 Reverse transcriptases (RTs) are typically thought of as molecular copy machines: they convert RNA template into DNA, with product length defined by template length. But DRT10 is different.