| Title |
Mechanistic studies of type III CRISPR-Cas activity regulation and characterization of associated translation-inhibiting effectors / |
| Translation of Title |
III tipo CRISPR-Cas aktyvumo reguliacijos mechanizmo tyrimai ir susijusių transliaciją slopinančių efektorių charakterizavimas. |
| Authors |
Mogila, Irmantas |
| DOI |
10.15388/vu.thesis.720 |
| Full Text |
|
| Pages |
196 |
| Keywords [eng] |
CRISPR-Cas ; FCS ; Csm ; RelE ; ribosome |
| Abstract [eng] |
CRISPR-Cas systems are widespread in prokaryotes providing adaptive protection against viral nucleic acids. Csm complexes of CRISPR-Cas type III-A systems recognize and cleave foreign RNA. This thesis aimed to supplement the established model of Csm activity with focus on their deactivation and determine the roles of ancillary proteins. Here, fluorescence correlation spectroscopy assays showed that despite rapid RNA shredding, the terminal RNA cleavage products remain bound to the complex for the mean duration of over an hour. Retained RNA stimulate DNase activity of Cas10 subunit of the complex and upon RNA release Cas10 is inhibited. Cas10 proteins also synthesize cyclic oligoadenylate molecules which activate ancillary CARF effectors. Sequence analysis shows that CARF proteins co-opted a mRNA interferase toxin called RelE giving rise to CARF-RelE fusion proteins Cami1. Biochemical and in vivo toxicity assays showed that Cami1 cleave mRNA and inhibit bacterial growth when activated by cyclic tetraadenylate. Ring nuclease activity of CARF domain allows to cleave the activator. Structural studies of Cami1 proteins revealed that they are recruited to the ribosomes via ribosomal stalk protein bL12. This is the first observation of a prokaryotic ribosome toxin hijacking the ribosome stalk to facilitate binding to the ribosome. |
| Dissertation Institution |
Vilniaus universitetas. |
| Type |
Doctoral thesis |
| Language |
English |
| Publication date |
2025 |
