Transcription regulation in Synechococcus elongatus and applications to synthetic Biology

Abstract

Cyanobacteria have evolved the ability to regulate transcription of their genes in cycles of 24 hours. These cycles, known as circadian rhythms, stem from an internal molecular clock that allows cyanobacteria to predict the onset of day and night. The cyanobacterial circadian clock – as studied in Synechococcus elongatus – consists of the three proteins KaiA, KaiB, and KaiC. By virtue of its intrinsic biochemical properties and its interactions with KaiA and KaiB, KaiC undergoes cycles of auto-phosphorylation and auto-dephosphorylation of exactly 24 hours. The cyanobacterial molecular clock being so simple, we sought tt to reconstitute it in E. coli as a device for synthetic biology. To this end, we assembled a small library of plasmids for the expression of the Kai proteins. Following screening of the library, we found one of the constructs was able to establish circadian oscillations of KaiC phosphorylation in E. coli. However, the core circadian clock does not regulate gene expression by itself. Instead, it relies on a cascade of additional factors (collectively known as the output pathway) to transmit the timing information to the RNA polymerase and the gene expression machinery. The terminal part of the output pathway relies on the master transcriptional regulator RpaA. Here, we show that RpaA can be used to activate the model circadian promoter P_kaiBC in E. coli. How RpaA regulates gene expression remains unclear. Thus, we sought to set up an in vitro transcription assay to shed light on RpaA’s mechanism of action. After testing six circadian promoters we found that promoter synpcc7942_0469 yields detectable amounts of transcript in vitro and suggest it can be used as a model circadian promoter to dissect RRpaA mechanism of action. Cyanobacteria are also interesting for their peculiar RNA polymerase. After resolving the Cryo-EM structure of S. elongatus RNA polymerase in collaboration with Prof. Murakami’s group at Pennsylvania State University, we found that its characteristically large SI3 domain may be flexible and protrude from the globular core of the enzyme. Interestingly, deletion of the SI3 tip from S. elongatus genome resulted in mild growth defects, and in vitro transcription experiments suggest the tip of the SI3 domain may play a role in promoter complex stability.