Anna Wang

Abstract:

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.

Abstract:

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.

Abstract:

Bacterial RNA Polymerases (RNAPs) bind to transcription initiation protein factors called σ-factors to start DNA sequence-specific transcription. Within σ-factors lies a highly conserved structural module, the ‘σ-finger’, a loop that that resides very close to the ‘heart’ of transcription, the active-centre of RNAP. The σ-finger is implicated in the pre-organisation of template DNA and the synthesis of the first short RNAs. The σ-finger also blocks entry of the nascent RNA to the RNA-exit channel of the RNAP and must be displaced to allow entry into transcription elongation. Despite structural studies, σ-finger conformational changes during late transcription initiation are still unknown. To uncover the dynamic conformational landscape and mechanism of the E. coli σ-finger during initial transcription and promoter escape, this thesis uses a new single-molecule FRET (smFRET) ruler. The results show that the σ-finger is displaced from its position inside the active site cleft, before promoter escape and after synthesis of RNA lengths that are highly dependent on the sequence of the promoter DNA used. Additionally, the chemical moiety at 5’-end of RNA, which are used in different modes of transcription, was also found to influence the point of σ-finger displacement. Real-time smFRET measurements revealed the presence of significant heterogeneity in the timing of σ-finger displacement and show that different initial conformations of the σ-finger are linked to significantly different kinetics in transcription initiation and promoter escape.

This thesis identifies different mechanisms of σ-finger displacement that influence the kinetics of initial transcription and have the potential to impact gene regulation in bacteria. Since archaeal and eukaryotic transcription systems contain σ-finger-like structural modules, these mechanisms may be general and apply to all kingdoms of life.