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Structural Studies of the Type III-A CRISPR Effector Complex in Staphylococcus epidermidis RP62a
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| 자료유형 | 학위논문 |
|---|---|
| 서명/저자사항 | Structural Studies of the Type III-A CRISPR Effector Complex in Staphylococcus epidermidis RP62a. |
| 개인저자 | Dorsey, Bryan W. |
| 단체저자명 | Northwestern University. Interdepartmental Biological Sciences (IBiS) Graduate Program. |
| 발행사항 | [S.l.]: Northwestern University., 2019. |
| 발행사항 | Ann Arbor: ProQuest Dissertations & Theses, 2019. |
| 형태사항 | 134 p. |
| 기본자료 저록 | Dissertations Abstracts International 81-02B. Dissertation Abstract International |
| ISBN | 9781085587679 |
| 학위논문주기 | Thesis (Ph.D.)--Northwestern University, 2019. |
| 일반주기 |
Source: Dissertations Abstracts International, Volume: 81-02, Section: B.
Advisor: Mondragon, Alfonso. |
| 이용제한사항 | This item must not be sold to any third party vendors.This item must not be added to any third party search indexes. |
| 요약 | Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types that vary widely in composition, target recognition, and overall mechanism. The main division of CRISPR-Cas systems occurs between Class 1 systems, which form multi-protein effector complexes, and Class 2 systems that utilize a single protein for defense. Specifically, Class 1 CRISPR-Cas systems are divided into three types, Type I, III, and IV, with most organisms harboring Type I or Type III systems. These systems utilize a CRISPR guide RNA (crRNA) that are composed of sequences that have been isolated from mobile genetic elements that have previously tried to infect the organism in order to identify the target for destruction.While much biochemical work has been done in both Type I and Type III systems, a far greater amount of structural studies have been performed on Type I systems when compared to Type III systems. In this context, we set out to expand the insights into the structural aspects of one of the Type III subtypes, Type III-A, CRISPR-Cas effector complexes. The Type III-A CRISPR-Cas system has been extensively studied biochemically in one particular organism, Staphylococcus epidermidis RP62a. We have focused our structural studies in this organism due to the wealth of this available biochemical data.Our experiments have allowed us to determine the crystal structures of the two most abundant components of the S. epidermidis RP62a effector complex, Csm2 and Csm3. The structure of Csm2 interestingly showed a hybrid topology of Type III-A and Type III-B homologs. This suggests that there may be more variability within effector complex subunit structures of closely related CRISPR systems, which may ultimately affect their organization and functional relevance within the context of the effector complex. The Csm3 structure was similar to previously solved homologs, but offered more details into regions that were missing in the other structures. This allowed us to provide the structural context for the myriad biochemically studied amino acids within Csm3, as well as to elucidate interesting conservational and biophysical organization within the protein.Additionally, we solved a 5.2 A resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an in vivo assembled effector subcomplex including the crRNA. The structure helps to clarify the quaternary architecture of Type III-A effector complexes by utilizing the Csm3 crystal structure to provide a more detailed view of how the subunits interact with each other in the complex, as well as providing details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes, as well as highlighting the overall similarities and differences with other Class 1 effector complexes. |
| 일반주제명 | Biophysics. Molecular biology. Biochemistry. |
| 언어 | 영어 |
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