9N9M

Cryo-EM structure of the dCas12f-gRNA-DNA complex (partial R-Loop)

Summary for 9N9M

• Entry DOI: 10.2210/pdb9n9m/pdb
• EMDB information: 49173
• Descriptor: Nuclease-deactivated Cas12f, DNA non-template strand, DNA template strand, ... (4 entities in total)
• Functional Keywords: dcas12f, partial r-loop, rna binding protein, rna binding protein-dna-rna complex, rna binding protein/dna/rna
• Biological source: Flagellimonas taeanensis; More
• Total number of polymer chains: 5
• Total formula weight: 152730.03

Authors

Chang, L.,Sternberg, S.H.,Xiao, R.,Hoffmann, F.T.,Wiegand, T.,Xie, D. (deposition date: 2025-02-11, release date: 2025-12-24, Last modification date: 2026-03-18)

Primary citation

Xiao, R.,Hoffmann, F.T.,Xie, D.,Wiegand, T.,Palmieri, A.I.,Sternberg, S.H.,Chang, L.
Structural basis of RNA-guided transcription by a dCas12f-sigma E -RNAP complex.
Nature, 2026

PubMed Abstract: In both natural and engineered biological systems, RNA-guided proteins have emerged as critical transcriptional regulators by modulating RNA polymerase (RNAP) and its associated factors. In bacteria, diverse 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 immunity. A distinct class of nuclease-dead Cas12f homologues (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σ), although the molecular basis has remained elusive. Here we reveal a new mode of RNA-guided transcription initiation by determining the cryo-electron microscopy structures of the dCas12f-σ system from Flagellimonas taeanensis. We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σ-RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σ-RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σ-dependent promoter recognition, these studies show that recognition of the -35 element is largely supplanted by CRISPR-Cas targeting, whereas 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 programmable transcriptional control.

PubMed: 41781609
DOI: 10.1038/s41586-026-10178-3

Experimental method

ELECTRON MICROSCOPY (3.38 Å)