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Hrle, Ajla (2014): The backbone of prokaryotic adaptive immunity: the Cas7 protein family. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



CRISPR/Cas is the prokaryotic adaptive immune response to viral invasion. Its mechanism is reminiscent of the eukaryotic RNA interference. The host actively incorporates short sequences from invading genetic elements (viruses or plasmids) into a region of its genome that is characterized by clustered regularly interspaced short palindromic repeats (CRISPRs) and a number of CRISPR-associated (cas) genes. The molecular memory of previous infections can be transcribed and processed into small RNAs (crRNAs) that guide a multiprotein–nucleic acid interference complex to recognize and cleave incoming foreign genetic material. Three pathways (I, II, III) are defined by their protein machinery and target specificity (DNA vs. RNA). In types I and III, the main protagonist of the interference complex is the Cas7 protein. Up to six copies of Cas7 constitute the complex’s main building block that assembles around the crRNA and provides a platform for protein interactions and target binding. During my PhD work, I solved the crystal structures of two Cas7 orthologs from different archaeal species, at 1.8 Å for Thermofilum pendens (Tp) Csc2 and at 2.37 Å for Meth- anopyrus kandleri (Mk) Csm3. The crystal structures of Mk Csm3 and Tp Csc2 were solved by experimental phasing and revealed a core RRM-like domain with a β1-α1-β2-β3-α2-β4 arrangement of secondary structure elements. The core is flanked by three peripheral domains that are defined by insertions within the core. Structural superposition of the RRM-like core domains of Mk Csm3 and Tp Csc2 with the representatives of other Cas families (5/6/7) revealed the highest homology beyond the RRM with a Cas7 family homolog. Thus I showed that Cas7 family proteins share equivalent insertions, forming homologous peripheral domains. Using the information obtained from structural data, I investigated the RNA binding properties Mk Csm3, Tp Csc2 and a Cas7 protein from subtype I-A, Thermoproteus tenax (Tt) Csa2. All orthologs bound RNA in a sequence-independent manner, according to their physiological function of spacer binding. Furthermore, a combined approach consisting of mutation analysis, UV-based protein–RNA crosslinking, mass spectrometry and fluorescence anisotropy mapped the RNA interacting regions to two structurally highly conserved positively charged surfaces. Taken together, this thesis describes a comprehensive structural study of the Cas7 family, defining the family’s structural features. These structural data from single proteins and the mapped RNA binding interfaces agree with protein–RNA interactions observed in the Escherichia coli interference complex.