ABSTRACT Bacterial sigma factors (σ) contain a highly conserved structural module, the ‘‘σ-finger’’, which forms a loop that protrudes towards the RNA polymerase (RNAP) active-centre in the open complex and has been implicated in pre-organisation of template DNA, abortive initiation of short RNAs, initiation pausing, and promoter escape. Here, we introduce a novel single-molecule FRET (smFRET) assay to monitor σ-finger motions during transcription initiation and promoter escape. We find that the σ-finger is displaced from its position inside the active site cleft before promoter escape, and after synthesis of RNAs with lengths that are highly dependent on the sequence of the promoter used. Real-time smFRET measurements reveal the presence of significant heterogeneity in the timing of finger displacement and show that different σ-finger conformations in single open transcription complexes are associated with substantially different kinetics in transcription initiation and promoter escape, potentially impacting gene regulation in bacteria.

Structured Illumination Microscopy, SIM, is one of the most powerful optical imaging methods available to visualize biological environments at subcellular resolution. Its limitations stem from a difficulty of imaging in multiple color channels at once, which reduces imaging speed. Furthermore, there is substantial experimental complexity in setting up SIM systems, preventing a widespread adoption. Here, we present Machine-learning Assisted, Interferometric Structured Illumination Microscopy, MAI-SIM, as an easy-to-implement method for live cell super-resolution imaging at high speed and in multiple colors. The instrument is based on an interferometer design in which illumination patterns are generated, rotated, and stepped in phase through movement of a single galvanometric mirror element. The design is robust, flexible, and works for all wavelengths. We complement the unique properties of the microscope with an open source machine-learning toolbox that permits real-time reconstructions to be performed, providing instant visualization of super-resolved images from live biological samples.

Sub-diffraction resolution, gentle sample illumination, and the possibility to image in multiple colors make Structured Illumination Microscopy (SIM) an imaging technique which is particularly well suited for live cell observations. Here, we present Machine learning Assisted Interferometric-SIM (MAI-SIM), an easy-to-implement method for high speed SIM imaging in multiple colors. The instrument is based on an interferometer design in which illumination patterns are generated, rotated, and stepped in phase through movement of a single galvanometric mirror element. The design is robust, flexible, and the pattern generation process works for all wavelengths. We complement the unique properties of interferometric SIM with a machine learning toolbox that is simple and efficient to use and is superior to existing methods for the reconstruction of super-resolved images recorded by the instrument. The framework permits real-time SIM reconstructions to be performed in multiple colors, providing the user with instant visualization of the super-resolved images. We demonstrate the capability of MAI-SIM on live biological samples and capture super-resolution images in multiple colors simultaneously over large fields of view. Finally, we embrace a fully open design philosophy to bring the advantages of MAI-SIM to as many users as possible and provide full details on system design and software.

The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3-0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes >1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.

Bacterial RNA Polymerases (RNAPs) bind to transcription initiation factors called σ factors to start promoter-specific transcription. Within the σ factor lies a highly conserved structural module, the “σ-finger”, which forms a loop that protrudes towards the RNAP active-center and that interacts with the template strand DNA. The close proximity of the σ-finger to the “heart” of transcription is implicated in the pre-organisation of the template strand DNA, in the synthesis of the first short RNAs, and in the pausing of transcription upon synthesis of a 6-mer RNA. However, the σ-finger also blocks entry of the nascent RNA to the RNAP exit channel and must be displaced during initial transcription as the first step in promoter escape. Despite recent structural studies, σ-finger conformational changes during late transcription initiation and promoter escape are still unknown. Here we report a novel single-molecule FRET ruler that monitors the conformational dynamics of the E.coli σ70-finger from the early stages of transcription initiation to promoter escape. Our results using the σ-finger FRET ruler on transcription complexes formed on a derivative of the lac promoter show that the σ-finger adopts three conformations, which interconvert with rates 0.01-1.5s−1. Intriguingly, we find the σ-finger first displaced in complexes trapped in synthesis of abortive transcripts up to a 10-mer RNA. This observation is likely driven by collision of the σ-finger with the 5’-end of the growing nascent RNA. Based on our results, we propose a new model describing the relation of the conformational changes in the σ-finger to the dynamics of transcription initiation and promoter escape. Archaeal and eukaryotic transcription initiation complexes also contain structural modules that block the RNAP exit channel, so it is therefore likely that this model applies to all kingdoms of life.