Transcription regulation by RpaA and RNAP in the circadian organism Synechococcus elongatus

Abstract

The cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) is a model organism for synthetic biology, circadian rhythms, and transcription regulation. Circadian clocks are valuable evolutionary tools that confer advantages to organisms through the matching of biochemical processes to anticipated environmental changes, and S. elongatus possesses the smallest and simplest observed timekeeping mechanism – the KaiABC complex. The cyclical changes in KaiC phosphorylation trigger signal transduction through output branches at key phases, which regulate the transcriptional profile of the cell. A main output branch involves the two-component system (TCS) SasA/CikA/RpaA, the latter of which has been termed the master regulator of S. elongatus’s transcription profile. Despite its key role in generating genome-wide transcription rhythms, RpaA’s mode of action is not understood. The transcriptional machinery of S. elongatus also deviates from the conserved prokaryotic make-up. The highly conserved Gre factors, crucial to transcription fidelity, are not observed in S. elongatus, suggesting alternative mechanisms for proofreading activity intrinsic to the RNA polymerase (RNAP). The processivity factor NusG appears to contain a sequence insertion spatially located in the proximity of upstream DNA. The polymerase also possesses features that deviate from typical multi-subunit RNAPs, including a split in the largest β’ subunit, the largest lineage-specific sequence insertion (SI3 domain), and a smaller diameter RNA-exit channel. In this work we investigated the molecular mechanism of transcription initiation regulation by RpaA. We demonstrated that RpaA recruits the RNAP to promoters by directly interacting with its ⍺ C-terminal domain. We showed that RpaA activity is sensitive to intracellular pH changes and redox state, suggesting additional degrees of circadian control over the cell’s transcriptional and metabolic profile. We also present evidence of RpaA’s association in complexes greater than dimers, forming filaments, and undergoing conformational shifts during DNA-binding. We have developed a model for the circadian mechanism that explains how two major promoter classes are activated and repressed in S. elongatus via RNAP-RpaA-DNA interactions. In another chapter we describe structure-activity relationship of the RNAP of S. elongatus, and the impact lineage-specific domains have on the catalytic activity of the enzyme. The largest subunit of cyanobacterial RNAP is divided into two polypeptides and contains the SI3 domain. We analysed the SI3’s role in transcription elongation and termination, as well as its impact on protein-protein interactions. We show that deletion of SI3 decreases pausing and termination efficiency. Due to the SI3’s location within the crucial trigger loop domain, this thesis also presents data showing the conservation of function of key residues in RNAchain elongation catalysis. Structural data show a narrowing of the RNA-exit channel by the 𝛽 subunit’s C-terminal region, which work presented here suggests acts in conjunction with NusG to prevent RNA hairpin-dependent RNAP pausing.