Biology subjects

Haack, D. B.

Publications and source records attributed to Haack, D. B..

4 recordsLinked to original sources

bioRxiv2026

Cryo-EM of a nucleotide-polymerizing ribozyme enables its predictive improvement

Ribozymes capable of self-replication from nucleotides would have been central to the hypothesized RNA World. The leading laboratory models for such molecules were converted from a class I ligase by in vitro evolution but then developed without 3D structures. Here, scaffolded cryo-EM of the substrate-free tC19Z RNA polymerase ribozyme at 3.1 [A] resolution shows how this conversion was achieved. An accessory domain evolved from random sequence grips the ancestral ligase through a loop-loop contact, a seam of magnesium ions, and a six-base stack, and rebuilds the ligases substrate binding site from different residues of its own. A previously unrecognized pairing, present before substrate binds, sequesters the 5' end that must otherwise pair with the template. Compensatory mutations to the ribozyme and template, designed to break this ectopic pairing, increase the extension rate. These results suggest that accelerating RNA structure determination may speed progress toward nucleotide-based self-replication.

biochemistry↗

bioRxiv2026

De novo design of RNA pseudoknots with deep learning

RNA design has been hindered by the limited accuracy of 3D structure prediction. Here, we show that intricate RNA structures can be generated with current deep learning tools through accurate de novo design of pseudoknot secondary structures. In an Eterna competition involving 57 pseudoknots, generative AI methods matched experienced human designers in solving most blind challenges, evaluated by single-nucleotide-resolution chemical mapping, compensatory mutagenesis, and cryogenic electron microscopy. AI-generated molecules with accurate secondary structures formed well-ordered 3D folds stabilized by noncanonical tertiary interactions not modeled during design. Success was guided by an RNet foundation model trained on prior chemical mapping data, suggesting that some difficult RNA design tasks may be tractable without first solving RNA 3D structure prediction.

biophysics↗

bioRxiv2025

Structural evidence for metal ion catalysis in the ribosome

Ribosomes synthesize proteins with an RNA-only active site across all domains of life, yet the details of the catalytic mechanism have remained elusive despite decades of high-resolution ribosome structures. Here, we provide structural evidence for the involvement of two metal ions in peptide bond formation, drawn from ribosome structures spanning bacteria, archaea, and eukaryotes. These metal ions reside in the peptidyl transferase center, one of them adjacent to a universally conserved stack of three base triples, reminiscent of the catalytic triplex in group I/II introns and the spliceosome, which catalyze pre-mRNA splicing. The second metal ion is positioned to stabilize the oxyanion of the tetrahedral intermediate. Metal ion catalysis thus emerges as a recurring mechanistic theme across the central dogma spanning protein synthesis, RNA splicing, and nucleic acid replication.

biochemistry↗

bioRxiv2024

Scaffold-enabled high-resolution cryo-EM structure determination of RNA

Cryo-EM structure determination of protein-free RNAs has remained difficult with most attempts yielding low to moderate resolution and lacking nucleotide-level detail. These difficulties are compounded for small RNAs as cryo-EM is inherently more difficult for lower molecular weight macromolecules. Here we present a strategy for fusing small RNAs to a group II intron that yields high resolution structures of the appended RNA, which we demonstrate with the 86-nucleotide thiamine pyrophosphate (TPP) riboswitch, and visualizing the riboswitch ligand binding pocket at 2.5 [A] resolution. We also determined the structure of the ligand-free apo state and observe that the aptamer domain of the riboswitch undergoes a large-scale conformational change upon ligand binding, illustrating how small molecule binding to an RNA can induce large effects on gene expression. This study both sets a new standard for cryo-EM riboswitch visualization and offers a versatile strategy applicable to a broad range of small to moderate-sized RNAs, which were previously intractable for high-resolution cryo-EM studies.

biochemistry↗