Rhythmic transcription in cyanobacteria

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 In our preliminary work we obtained first two structures of cyanobacterial initiation and elongation complexes (unpublished).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.