Control of a type III-Dv CRISPR-Cas system by the transcription factor RpaB and interaction of its transcribed leader with the DEAD-box RNA helicase CrhR

CRISPR-Cas systems in bacteria and archaea provide powerful defense against phages and other foreign genetic elements. The principles of CRISPR-Cas activity are well understood, but less is known about how their expression is regulated. The cyanobacterium Synechocystis sp. PCC 6803 encodes three different CRISPR-Cas systems. The expression of one of them, a type III-Dv system, responds to changes in environmental conditions, such as nitrogen starvation or varying light intensities. Here, we found that the promoter of the respective six-gene cas operon is controlled by the light- and redox-responsive transcription factor RpaB. RpaB binds to an HLR1 motif located 53 to 70 nt upstream of the transcription start site, resulting in transcriptional activation at low light intensities. However, the strong promoter driving transcription of the cognate repeat-spacer array is not controlled by RpaB. Instead, we found that the 125 nt transcribed leader is bound by the redox-sensitive RNA helicase CrhR. Cross-linking coupled to mass spectrometry analysis revealed six residues involved in the CrhR-RNA interaction. Of these, L103, F104, H225, and C371 were predicted to be on the surface of a dimeric CrhR model, while C184 was not and P443 in the very C-terminal region could not be assigned to a structural element. These results show that the expression of the CRISPR-Cas system is linked to the redox status of the photosynthetic cyanobacterial cell at two different levels. While RpaB affects transcription, CrhR interacts with the transcribed leader post-transcription. These results highlight the complex interplay between a CRISPR-Cas system and its host cell. ### Competing Interest Statement The authors have declared no competing interest.