Rhythmic transcription in cyanobacteria

Life on Earth depends on light and warmth of the Sun. One of the largest group of organisms on Earth is cyanobacteria who use energy of sun to fix cardon dioxide and produce oxygen during photosynthesis. Cyanobacteria have evolved the ability to regulate activity of their genes in cycles of 24 hours. These cycles are based not only on actual day/night cycles, but also supported by internal circadian molecular clock that allows cyanobacteria to predict the onset of day and night.
Light-dependent and circadian- information is collated by RNA polymerase (RNAP) and transformed into rhythmic pattern of gene expression. How this information is deciphered by RNAP is not known.
We aim to characterise domains of RNAP, transcription factors and DNA regulatory elements involved in circadian activation/repression of genes by using bioinformatics, biochemistry, proteomics and Cryo-EM microscopy. In preliminary work we developed tractable in vitro experimental system of transcription and circadian regulation, and obtained first two structures of cyanobacterial RNAP complexes, supported by biochemical and proteomics data.
This project will generate fundamental knowledge of cyanobacteria, who are the main contributors in global carbon cycle. Cyanobacteria currently gaining popularity as carbon neutral producers. Our research will inform genetic and metabolic engineering of industrial strains. In the future cyanobacterial circadian clock transplanted into industrial microorganisms may serve as attractive mechanism to schedule complex biosynthetic tasks.

Cyanobacteria are key primary producers, who use sunlight to fix bulk of carbon dioxide on Earth and produce oxygen. Transcription of their genes quickly adjusts to light, repressed during the darkness/night by alarmone ppGpp. In addition, cyanobacteria are the only prokaryotes possessing circadian clock ticking independently from actual light and allowing to predict onset of day and night. Transcription of ~90% of genes in model species Synechococcus elongatus 7942 are rhythmically regulated, and their activity is scheduled to peak with 24h periodicity at particular time of the day.
Cyanobacterial RNA polymerase (RNAP) is the main target of light/dark and circadian regulation. How RNAP may integrate light and circadian information is not known.
In our preliminary work we obtained first two structures of cyanobacterial initiation and elongation complexes (unpublished). They show a number of unique features: atypical huge SI3 insertion into Trigger Loop makes direct contact with initiation sigma factor and reaches NusG in elongation complex; one of the two ppGpp binding sites is missing; NusG factor has specific insert which contacts downstream DNA. To investigate how circadian signals regulate transcription initiation, we reconstructed phosphoryl transfer path from the clock to DNA-binding master regulator in vitro.
In the project we will investigate role of unique SI3 interactions in promoter complex formation; specificity of sigma factors and its role in sigma cascade; regulation of initiation by ppGpp, mechanism of circadian factors regulation of transcription; and role of SI3 and NusG in elongation and pausing.
This project will generate fundamental knowledge of cyanobacteria, the major contributors in global carbon cycle.