Structural basis of RNA-guided transcription by a dCas12f-σ ^E -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 factors ^1-5 . 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 ^1,6,7 . Intriguingly, a distinct class of nuclease-dead Cas12f homologs (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σ ^E ) ⁸ , 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-σ ^E system from Flagellimonas taeanensis . We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σ ^E -RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σ ^E -RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σ ^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 programmable transcriptional control.