Characterization of the CRISPR-Cas subtype I-B proteins Cas6b and Cas8b of Methanococcus maripaludis C5
The CRISPR-Cas system is an adaptive immune system found in archaea and bacteria to defend themselves against mobile genetic elements (e.g. phages). The system employs base complementarity of small RNA species (crRNAs) to target the foreign nucleic acids for degradation. The hallmark of the syste...
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|Zusammenfassung:||The CRISPR-Cas system is an adaptive immune system found in archaea and bacteria to defend themselves against mobile genetic elements (e.g. phages). The system employs base complementarity of small RNA species (crRNAs) to target the foreign nucleic acids for degradation. The hallmark of the system is the CRISPR array or locus, which is composed of repetitive DNA sequences (repeats) that are interspersed by unique sequences (spacers). Spacer sequences can be derived from earlier encounters with viruses and, as part of the crRNAs, confer the base complementarity during a reoccurring attack. During the ongoing battle between prokaryotes and viruses diverse CRISPR-Cas systems evolved into three main types that are further subdivided. This thesis shows the first characterization of a subtype I-B CRISPR-Cas system. RNA-Seq data proved the in vivo activity of this CRISPR-Cas system in Methanococcus maripaludis C5. The data further revealed that the crRNAs are always composed of a complete spacer sequence flanked by an 8 nt 5' repeat tag and a 2 nt 3' repeat tag. Eigth cas genes were identified for M. maripaludis. Two Cas proteins, Cas8b and an annotated hypothetical protein were characterized in more detail. The hypothetical protein was shown to be the endoribonuclease responsible for the single-turnover catalysis of precursor crRNA into mature crRNA and was termed Cas6b. The reaction performed by Cas6b yields the 8 nt 5' terminal tag of the mature crRNAs. Despite sharing only low sequence identity of 11 %, the two Cas6 proteins of M. maripaludis and Pyrococcus furiosus could be well aligned using a structural model of Cas6b and the crystal structure of P. furiosus Cas6. Cas6b mutant analysis was used to determine four amino acid residues (lysine 30, histidine 38, histidine 40 and tyrosine 47) that comprise the catalytic site of Cas6b. The RNA binding properties of Cas6b were determined and showed a dimerization upon binding to a non-cleavable substrate. Further analyses including RNA crosslinking experiments followed by mass spectrometry identified a methionine residue (M185) that tightly coordinated to a uridine (U15) of the repeat sequence. Cas6b activity assays employing differently structured repeat variants of M. maripaludis and a 37 nt repeat sequence of Clostridium thermocellum could show, that the processing reaction performed by Cas6b does not recognize a secondary structure of the substrate. In addition to the verification to the in vivo activity of the CRISPR-Cas system, the RNA-Seq data also revealed a varying abundance pattern of crRNAs. To assess the crRNA abundance a experimental procedure was designed, which was aimed to analyse the influence of spacer sequences on a) the processing by Cas6 and b) the stability of crRNAs. With the help of this global approach influences of the spacer length and spacer sequence on the crRNA maturation and in vitro stability were recognized. In this context, future experiments will also determine further possible influences on crRNA abundance including i) crRNA loading into the Cas protein interference complex (Cascade) and ii) possible regulatory effects in terms of crRNA utilization dependent regulation. The characterization of the subtype-specific protein Cas8b revealed a splitting of the recombinant protein into two defined fragments. The exact point of cleavage was determined by Edman sequencing and provides evidence for a proteolytic cleavage of the full-length protein (either autocatalytically or by a protease). Other CRISPR-Cas subtypes were reported to contain two proteins serving as small and big subunit of the interference complex Cascade. For subtype I-B on the other hand Cas8b was found to be the only equivalent to these two proteins and it was proposed that the identified cleavage generates the large and small Cascade subunit. A biochemical analysis of Cas8b with respect to its putative roles during CRISPR-Cas immunity showed an unspecific binding to nucleic acids while no nucleolytic cleavage was observed. Possible functions of Cas8b are discussed and future studies will focus on the analysis of the protein functions in the context of a complete Cascade.|