Gene machines

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.