The “Christmas tree” structures resolved by Miller in the 1980s provided the first detailed electron micrographs of E. coli rRNA operons, revealing densely packed RNA polymerases with nascent transcripts. Under rapid growth conditions, up to 100 RNA polymerase complexes can transcribe a single operon simultaneously, creating an exceptionally dense transcriptional landscape. However, how the seven ∼5 kb operons, each with a contour length of ∼1.7 μm, are compacted within a 1–2 μm cell remains unresolved. Also, Miller’s pioneering work captured only a static ex vivo snapshot of rRNA transcription. Real-time visualization of these processes has remained a significant challenge since conventional microscopy lacks the spatial and temporal resolution necessary to resolve operon organization and the co-transcriptional assembly of ribosomes. MINFLUX nanoscopy, with sub-5 nm spatial resolution, provides a powerful approach to interrogate these mechanisms. Its ability to resolve RNA polymerase complexes spaced ∼20 nm apart enables direct mapping of polymerase distributions along rRNA operons. With nanometer-precision 3D localization, we defined operon ultrastructure and nucleoid positioning in fixed cells. We also applied this approach to different E. coli strains and under diverse growth conditions to investigate transcriptional responses in varied physiological contexts. In addition, MINFLUX provides spatiotemporal resolution for single-fluorophore tracking, enabling in vivo visualization of dynamic ribosome assembly. We labeled the methyltransferases RlmC and RlmD, along with their mutant variants engineered to covalently trap these enzymes at intermediate states during ribosome assembly. This strategy allowed for direct observation of the interactions between rRNA and assembly factors. Our work applies MINFLUX nanoscopy to address critical gaps in our understanding of nucleoid organization, transcriptional coordination, and the molecular mechanisms of ribosome assembly in bacteria system. Our preliminary results revealed how rRNA transcription and nucleoid architecture are coupled to bacterial growth regulation.
Chen et al. (Sun,) studied this question.