Abstract
The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated proteins) is one of the important known immune mechanisms in archaea and bacteria. This adaptive immune system degrades invading genetic elements and protects the cell. Amongst 3 main types I, II and III of CRISPR system, two types (I and III) are found in archaea. However, in Sulfolobus species, subtypes IA, I-D, and III-B, III-D and rarely III-A are found. The model organism used for interference and structural studies is S. islandicus REY15A which carries subtypes I-A and III-B (α and β). Besides CRISPR ribonucleoprotein complex which is involved directly in defense, there are some less- known parts of the system including CPBs (CRISPR repeat-binding proteins) which are suggested to play a role in transcription.
In the first part of my thesis, I provide a brief introduction to archaea and viruses that infect archaeal species followed by an introduction to CRISPR, including a description of the different types and subtypes, the role of the seed sequence, PAM and general mode of action of CRISPR. I also provide a brief summary of CRISPR-binding proteins.
In the second part, I present the results obtained from experimental work performed to investigate the effect of imperfect base-paring of crRNA-protospacer and various PAMs on CRISPR targeting activity of a subtype I-A subfamily in S. islandicus REY15A. The results showed the existence of two important crRNA-protospacer annealing regions along the first spacer of locus 2. We demonstrated that the presence of more than 3 mutations in this region can reduce interference, significantly. We also showed that NC and CN PAMs except CG can reduce the interference more profoundly than the other PAMs. Based on the orientation of the designed plasmid constructs and upstream promoter, we inferred that the obtained results are cause by subtype I-A interference system. The subtype III-B deletion strain was also used as a host which confirmed the involvement of to the subtype I-A CRISPR system as the main operating interference system.
In the third part, I present the results from finding new archaeal CBP homologues, DNA-binding activity of and its dual-domain variants, purification of CBP1 to remove all bound unspecific DNA and resolution of the structure of first domain by NMR analysis. Furthermore, results from purification of the CBP1- repeat DNA complex and its crystallization for the X-ray crystallography analysis are also presented. I found two new archaeal CBP homologues in public sequence databases. The tri-partite protein, CBP1 was selected to construct dual domain variants from its domains R1R2; R2R3 and R1R3 to study the influence of each dual domain variant on DNA- binding activity. Dual domain variant R1R 2 showed higher affinities to repeat DNA than the other two dual domain variants. Bioinformatics analyses predicted the tertiary structure of CBP1 and revealed that CBP1 has three HTH internal domains with two flexible linkers. The NMR backbone results showed that the protein is folded and the NMR structure of the protein also indicated a well-structured first domain which is stabilized by hydrophobic side chain interactions. However, the second domain is partially structured while the third domain in not structured which could be because of lack of information. I also purified CBP1 bound to DNA repeat specifically in order to crystalize it. I succeeded in getting the single crystals; however more optimization is required in order to obtain a big enough crystal to get diffracted by the X-rays. Since the HTH structure and DNA-binding activity is a common feature of transcriptional regulators, we therefore suggest that this protein could potentially play a role in transcription.
In the first part of my thesis, I provide a brief introduction to archaea and viruses that infect archaeal species followed by an introduction to CRISPR, including a description of the different types and subtypes, the role of the seed sequence, PAM and general mode of action of CRISPR. I also provide a brief summary of CRISPR-binding proteins.
In the second part, I present the results obtained from experimental work performed to investigate the effect of imperfect base-paring of crRNA-protospacer and various PAMs on CRISPR targeting activity of a subtype I-A subfamily in S. islandicus REY15A. The results showed the existence of two important crRNA-protospacer annealing regions along the first spacer of locus 2. We demonstrated that the presence of more than 3 mutations in this region can reduce interference, significantly. We also showed that NC and CN PAMs except CG can reduce the interference more profoundly than the other PAMs. Based on the orientation of the designed plasmid constructs and upstream promoter, we inferred that the obtained results are cause by subtype I-A interference system. The subtype III-B deletion strain was also used as a host which confirmed the involvement of to the subtype I-A CRISPR system as the main operating interference system.
In the third part, I present the results from finding new archaeal CBP homologues, DNA-binding activity of and its dual-domain variants, purification of CBP1 to remove all bound unspecific DNA and resolution of the structure of first domain by NMR analysis. Furthermore, results from purification of the CBP1- repeat DNA complex and its crystallization for the X-ray crystallography analysis are also presented. I found two new archaeal CBP homologues in public sequence databases. The tri-partite protein, CBP1 was selected to construct dual domain variants from its domains R1R2; R2R3 and R1R3 to study the influence of each dual domain variant on DNA- binding activity. Dual domain variant R1R 2 showed higher affinities to repeat DNA than the other two dual domain variants. Bioinformatics analyses predicted the tertiary structure of CBP1 and revealed that CBP1 has three HTH internal domains with two flexible linkers. The NMR backbone results showed that the protein is folded and the NMR structure of the protein also indicated a well-structured first domain which is stabilized by hydrophobic side chain interactions. However, the second domain is partially structured while the third domain in not structured which could be because of lack of information. I also purified CBP1 bound to DNA repeat specifically in order to crystalize it. I succeeded in getting the single crystals; however more optimization is required in order to obtain a big enough crystal to get diffracted by the X-rays. Since the HTH structure and DNA-binding activity is a common feature of transcriptional regulators, we therefore suggest that this protein could potentially play a role in transcription.
| Original language | English |
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| Publisher | Department of Biology, Faculty of Science, University of Copenhagen |
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| Number of pages | 175 |
| Publication status | Published - 2015 |