An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3.
ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR) chromosomal loci found in prokaryotes provide an adaptive immune system against bacteriophages and plasmids. CRISPR-specific endoRNases produce short RNA molecules (crRNAs) from CRISPR transcripts, which harbor sequences complementary to invasive nucleic acid elements and ensure their selective targeting by CRISPR-associated (Cas) proteins. The extreme sequence divergence of CRISPR-specific endoRNases and their RNA substrates has obscured homology-based comparison of RNA recognition and cleavage mechanisms. Here, we show that Cse3 type CRISPR-specific endoRNases bind a hairpin structure and residues downstream of the cleavage site within the repetitive segment of cognate CRISPR RNA. Cocrystal structures of Cse3-RNA complexes reveal an RNA-induced conformational change in the enzyme active site that aligns the RNA strand for site-specific cleavage. These studies provide insight into a catalytically essential RNA recognition mechanism by a large class of CRISPR-related endoRNases.
Article: Function and Regulation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR Associated (Cas) Systems.[show abstract] [hide abstract]
ABSTRACT: Phages are the most abundant biological entities on earth and pose a constant challenge to their bacterial hosts. Thus, bacteria have evolved numerous 'innate' mechanisms of defense against phage, such as abortive infection or restriction/modification systems. In contrast, the clustered regularly interspaced short palindromic repeats (CRISPR) systems provide acquired, yet heritable, sequence-specific 'adaptive' immunity against phage and other horizontally-acquired elements, such as plasmids. Resistance is acquired following viral infection or plasmid uptake when a short sequence of the foreign genome is added to the CRISPR array. CRISPRs are then transcribed and processed, generally by CRISPR associated (Cas) proteins, into short interfering RNAs (crRNAs), which form part of a ribonucleoprotein complex. This complex guides the crRNA to the complementary invading nucleic acid and targets this for degradation. Recently, there have been rapid advances in our understanding of CRISPR/Cas systems. In this review, we will present the current model(s) of the molecular events involved in both the acquisition of immunity and interference stages and will also address recent progress in our knowledge of the regulation of CRISPR/Cas systems.Viruses 01/2012; 4(10):2291-311. · 1.50 Impact Factor
Article: Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site.[show abstract] [hide abstract]
ABSTRACT: Precise RNA processing is fundamental to all small RNA-mediated interference pathways. In prokaryotes, clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode small CRISPR RNAs (crRNAs) that protect against invasive genetic elements by antisense targeting. CRISPR loci are transcribed as a long precursor that is cleaved within repeat sequences by CRISPR-associated (Cas) proteins. In many organisms, this primary processing generates crRNA intermediates that are subject to additional nucleolytic trimming to render mature crRNAs of specific lengths. The molecular mechanisms underlying this maturation event remain poorly understood. Here, we defined the genetic requirements for crRNA primary processing and maturation in Staphylococcus epidermidis. We show that changes in the position of the primary processing site result in extended or diminished maturation to generate mature crRNAs of constant length. These results indicate that crRNA maturation occurs by a ruler mechanism anchored at the primary processing site. We also show that maturation is mediated by specific cas genes distinct from those genes involved in primary processing, showing that this event is directed by CRISPR/Cas loci.Proceedings of the National Academy of Sciences 12/2011; 108(52):21218-22. · 9.68 Impact Factor