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Structural studies of CRISPR-associated proteins

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JudithReeksPhDThesis.pdf (136.0Mb)
Date
29/11/2013
Author
Reeks, Judith
Supervisor
Naismith, Jim
Keywords
X-ray crystallography
CRISPR/Cas
S. solfataricus
FaNaC
ASIC
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Abstract
Clustered regularly interspaced short palindromic repeats (CRISPRs) act to prevent viral infection and horizontal gene transfer in prokaryotes. The genomic CRISPR array contains short sequences (“spacers”) that are derived from foreign genetic elements. The CRISPR array is transcribed and processed into CRISPR RNAs (crRNAs) used in the sequence-specific degradation of foreign nucleic acids. This process is called interference and is mediated by CRISPR-associated (Cas) proteins. This thesis has focused on the structural and functional characterisation of four Cas proteins from the CRISPR/Cas system of Sulfolobus solfataricus. The crystal structure of Cmr7 (Sso1725), a Sulfolobales-specific subunit of the ssRNA-degrading CMR complex, allowed for the identification of a putative protein-binding site, though no specific function could be ascribed to the protein. Cas6 (Sso1437) is the enzyme responsible for crRNA maturation and the characterisation of this protein allowed for the molecular rationalisation of its atypical RNA cleavage mechanism. Csa5 and Cas8a2 are subunits of the aCascade complex that targets dsDNA. Csa5 (Sso1398) was shown to have a putative role in R-loop stabilisation during interference while the role of Cas8a2 (Sso1401) was not determined. The structures of these two proteins were used to define relationships between the subunits of interference complexes from various CRISPR/Cas systems. A second aspect of this work has been the expression and purification of eukaryotic ion channels for structural studies. The acid sensing ion channel (ASIC) and FMRFamide-gated sodium channel (FaNaC) are gated ion channels with unknown mechanisms of channel activation. These ion channels must be expressed in eukaryotic systems and so human embryonic kidney (HEK) cells and baculovirus-insect cell expression systems were developed to express ASIC and FaNaC constructs. The expression and purification protocols have been optimised to allow for the preparation of soluble protein that will in future be used for crystallography and electron paramagnetic resonance (EPR) studies.
Type
Thesis, PhD Doctor of Philosophy
Collections
  • Biomedical Sciences Research Complex (BSRC) Theses
  • Chemistry Theses
Description of related resources
Reeks , J , Sokolowski , R D , Graham , S , Liu , H , Naismith , J H & White , M F 2013 , ' Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing ' Biochemical Journal , vol 452 , no. 2 , pp. 223-230
Reeks , J A , Naismith , J & White , M F 2013 , ' CRISPR interference : a structural perspective ' Biochemical Journal , vol 453 , no. 2 , pp. 155-166
Reeks , J A , Graham , S , Anderson , L , Liu , H , White , M F & Naismith , J 2013 , ' Structure of the archaeal Cascade subunit Csa5 : Relating the small subunits of CRISPR effector complexes ' RNA Biology , vol 10 , no. 5 , pp. 762-769
Zhang , J , Rouillon , C , Kerou , M , Reeks , J , Brugger , K , Graham , S , Reimann , J , Cannone , G , Liu , H , Albers , S-V , Naismith , J H , Spagnolo , L & White , M F 2012 , ' Structure and mechanism of the CMR complex for CRISPR-Mediated antiviral immunity ' Molecular Cell , vol 45 , no. 3 , pp. 303-313
URI
http://hdl.handle.net/10023/3965
http://hdl.handle.net/10023/3954
http://hdl.handle.net/10023/3863
http://hdl.handle.net/10023/3550
http://hdl.handle.net/10023/5991

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