FUNCTIONAL ANALYSIS OF SUBUNITS OF THE RNA-GUIDED CRISPR IMMUNE SYSTEM IN THERMOTOGA MARITIMA

clustered regularly interspaced short palindromic repeats (CRISPR);Cmr;Cmr5;repeat- associated mysterious protein (RAMP);RNA interference;nuclease;not listed

Kuo, Fang TingBailey, Scott ;

Johns Hopkins University

关键词: clustered regularly interspaced short palindromic repeats (CRISPR);    Cmr;    Cmr5;    repeat- associated mysterious protein (RAMP);    RNA interference;    nuclease;    not listed;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39237/KUO-THESIS-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The CRISPR (clustered regularly interspaced short palindromic repeats) system is an adaptive immune system utilized by prokaryotes against invasive nucleic acid.CRISPR-derived RNA (crRNA) and CRISPR associated (CAS) proteins form the effector complexes that degrade foreign genetic elements and serve as the basis of a prokaryotic defense system. The CMR complex, which is composed of either six or seven protein subunits (Cmr1-6 or 1-7), is unique among all types of CRISPR/CAS systems because it targets RNA rather than DNA for degradation. So far, three different mechanisms have been proposed in Pyrococcus furiosus, Sulfolobus solfataricus, and Thermus thermophilus. Here, we are able to recombinantly express and purify the individual Cmr subunits of Thermotoga maritima, form the CMR complex with a short crRNA in vitro, and demonstrate the nuclease activity of the complex. Using pull-down assays we have mapped the interactions between Cmr protein subunits, which is consistent with the electron microscopy studies of CMR complex in T. thermophilus and P. furiosus. We have also begun to elucidate the biochemical roles of the Cmr5 subunit in the CMR complex of T. maritima. From mutational analysis, we conclude that Cmr5 may play a role in the processivity of the CMR nuclease activity and is critical to forming a stable CMR complex.