Structural and Functional Characterization of T.thermophilus CasE

  • Author / Creator
    Gesner, Emily
  • Powerful mechanisms of genetic interference in both unicellular and multicellular organisms are based on the sequence-directed targeting of DNA or RNA by small effector RNAs. In many bacteria and almost all archaea, RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci are involved in an adaptable and heritable gene-silencing pathway. Resistance to phage infection is conferred by the incorporation of short invading DNA sequences into the prokaryotic genome as CRISPR spacer elements separated by short repeat sequences. A central aspect to this pathway is the processing of a long primary transcript (pre-crRNA) containing these repeats by crRNA endonucleases to generate the mature effector RNAs that interfere with phage or plasmid gene expression. Here we describe a structural and functional analysis of the CasE endonuclease of T. thermophilus a member of the Ecoli CRISPR sub-type. High resolution X-ray structures of CasE bound to repeat RNAs model both the pre-and post-cleavage complexes associated with processing the pre-crRNA. These structures establish the molecular basis of a specific CRISPR RNA recognition and suggest the mechanism for generation of effector RNAs responsible for gene-silencing.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Biochemistry
  • Supervisor / co-supervisor and their department(s)
    • MacMillan, Andrew (Department of Biochemistry)
  • Examining committee members and their departments
    • Unrau, P (Molecular Biology and Biochemistry, SFU)
    • MacMillan, Andrew (Department of Biochemistry)
    • Bleackley, R Chris (Department of Biochemistry)
    • Owttrim, G (Department of Biological Sciences)
    • Glover, M (Department of Biochemistry)