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  • 1
    Publication Date: 2009-08-15
    Description: Transfer RNAs are among the most ubiquitous molecules in cells, central to decoding information from messenger RNAs on translating ribosomes. In eukaryotic cells, tRNAs are actively transported from their site of synthesis in the nucleus to their site of function in the cytosol. This is mediated by a dedicated nucleo-cytoplasmic transport factor of the karyopherin-beta family (Xpot, also known as Los1 in Saccharomyces cerevisiae). Here we report the 3.2 A resolution structure of Schizosaccharomyces pombe Xpot in complex with tRNA and RanGTP, and the 3.1 A structure of unbound Xpot, revealing both nuclear and cytosolic snapshots of this transport factor. Xpot undergoes a large conformational change on binding cargo, wrapping around the tRNA and, in particular, binding to the tRNA 5' and 3' ends. The binding mode explains how Xpot can recognize all mature tRNAs in the cell and yet distinguish them from those that have not been properly processed, thus coupling tRNA export to quality control.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Cook, Atlanta G -- Fukuhara, Noemi -- Jinek, Martin -- Conti, Elena -- England -- Nature. 2009 Sep 3;461(7260):60-5. doi: 10.1038/nature08394.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Structural Cell Biology, MPI for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19680239" target="_blank"〉PubMed〈/a〉
    Keywords: Binding Sites ; Cell Nucleus/*metabolism ; Crystallography, X-Ray ; Cytosol/*metabolism ; GTPase-Activating Proteins/chemistry/metabolism ; Models, Molecular ; Nuclear Pore Complex Proteins/*chemistry/*metabolism ; Protein Binding ; Protein Conformation ; *RNA Transport ; RNA, Fungal/chemistry/genetics/metabolism ; RNA, Transfer/chemistry/genetics/*metabolism ; RNA, Transfer, Phe/chemistry/genetics/metabolism ; Saccharomyces cerevisiae Proteins/chemistry/metabolism ; Schizosaccharomyces pombe Proteins/*chemistry/*metabolism ; Substrate Specificity ; ran GTP-Binding Protein/chemistry/metabolism
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 2
    Publication Date: 2009-01-23
    Description: In eukaryotes, small non-coding RNAs regulate gene expression, helping to control cellular metabolism, growth and differentiation, to maintain genome integrity, and to combat viruses and mobile genetic elements. These pathways involve two specialized ribonucleases that control the production and function of small regulatory RNAs. The enzyme Dicer cleaves double-stranded RNA precursors, generating short interfering RNAs and microRNAs in the cytoplasm. These small RNAs are transferred to Argonaute proteins, which guide the sequence-specific silencing of messenger RNAs that contain complementary sequences by either enzymatically cleaving the mRNA or repressing its translation. The molecular structures of Dicer and the Argonaute proteins, free and bound to small RNAs, have offered exciting insights into the molecular mechanisms that are central to RNA silencing pathways.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Jinek, Martin -- Doudna, Jennifer A -- R01 GM073794/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2009 Jan 22;457(7228):405-12. doi: 10.1038/nature07755.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19158786" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Humans ; MicroRNAs/*biosynthesis/genetics/*metabolism ; RNA Interference/*physiology ; RNA, Small Interfering/*biosynthesis/genetics/*metabolism ; RNA-Induced Silencing Complex/metabolism ; Ribonuclease III/metabolism
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    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 3
    Publication Date: 2010-09-11
    Description: Many bacteria and archaea contain clustered regularly interspaced short palindromic repeats (CRISPRs) that confer resistance to invasive genetic elements. Central to this immune system is the production of CRISPR-derived RNAs (crRNAs) after transcription of the CRISPR locus. Here, we identify the endoribonuclease (Csy4) responsible for CRISPR transcript (pre-crRNA) processing in Pseudomonas aeruginosa. A 1.8 angstrom crystal structure of Csy4 bound to its cognate RNA reveals that Csy4 makes sequence-specific interactions in the major groove of the crRNA repeat stem-loop. Together with electrostatic contacts to the phosphate backbone, these enable Csy4 to bind selectively and cleave pre-crRNAs using phylogenetically conserved serine and histidine residues in the active site. The RNA recognition mechanism identified here explains sequence- and structure-specific processing by a large family of CRISPR-specific endoribonucleases.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133607/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133607/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Haurwitz, Rachel E -- Jinek, Martin -- Wiedenheft, Blake -- Zhou, Kaihong -- Doudna, Jennifer A -- 5 T32 GM08295/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- New York, N.Y. -- Science. 2010 Sep 10;329(5997):1355-8. doi: 10.1126/science.1192272.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20829488" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Substitution ; Bacterial Proteins/*chemistry/*metabolism ; Base Pairing ; Base Sequence ; CRISPR-Associated Proteins ; Crystallization ; Crystallography, X-Ray ; Endoribonucleases/*chemistry/*metabolism ; Genes, Bacterial ; Hydrogen Bonding ; Models, Molecular ; Nucleic Acid Conformation ; Protein Conformation ; Protein Structure, Tertiary ; Pseudomonas aeruginosa/*enzymology/*genetics ; *RNA Processing, Post-Transcriptional ; RNA, Bacterial/chemistry/genetics/*metabolism ; *Repetitive Sequences, Nucleic Acid ; Static Electricity
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 4
    Publication Date: 2014-01-31
    Description: The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106473/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106473/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Sternberg, Samuel H -- Redding, Sy -- Jinek, Martin -- Greene, Eric C -- Doudna, Jennifer A -- GM074739/GM/NIGMS NIH HHS/ -- R01 GM073794/GM/NIGMS NIH HHS/ -- R01 GM074739/GM/NIGMS NIH HHS/ -- T32 GM066698/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2014 Mar 6;507(7490):62-7. doi: 10.1038/nature13011. Epub 2014 Jan 29.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉1] Department of Chemistry, University of California, Berkeley, California 94720, USA [2]. ; 1] Department of Chemistry, Columbia University, New York, New York 10032, USA [2]. ; 1] Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA [2] Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland. ; Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University, New York, New York 10032, USA. ; 1] Department of Chemistry, University of California, Berkeley, California 94720, USA [2] Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA [3] Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA [4] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/24476820" target="_blank"〉PubMed〈/a〉
    Keywords: Apoenzymes/metabolism ; *Base Pairing ; Base Sequence ; Biocatalysis ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/chemistry/genetics/metabolism ; *DNA Cleavage ; Diffusion ; Endonucleases/*metabolism ; Enzyme Activation ; Genetic Engineering/methods ; Genome/genetics ; Nucleic Acid Denaturation ; Nucleic Acid Heteroduplexes/chemistry/genetics/metabolism ; Nucleotide Motifs ; RNA/chemistry/*genetics/metabolism ; Streptococcus pyogenes/enzymology/immunology ; Substrate Specificity ; Thermodynamics
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 5
    Publication Date: 2014-08-01
    Description: The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176945/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176945/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Anders, Carolin -- Niewoehner, Ole -- Duerst, Alessia -- Jinek, Martin -- 337284/European Research Council/International -- England -- Nature. 2014 Sep 25;513(7519):569-73. doi: 10.1038/nature13579. Epub 2014 Jul 27.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25079318" target="_blank"〉PubMed〈/a〉
    Keywords: Arginine/genetics/metabolism ; *Base Pairing ; Base Sequence ; CRISPR-Associated Proteins/*metabolism ; Crystallography, X-Ray ; DNA/*chemistry/genetics/*metabolism ; Endonucleases/*metabolism ; Models, Molecular ; Nucleic Acid Denaturation ; *Nucleotide Motifs ; Protein Conformation ; RNA, Guide/chemistry/genetics/metabolism ; Streptococcus pyogenes/*enzymology ; Substrate Specificity
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 6
    Publication Date: 2012-06-30
    Description: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Jinek, Martin -- Chylinski, Krzysztof -- Fonfara, Ines -- Hauer, Michael -- Doudna, Jennifer A -- Charpentier, Emmanuelle -- Howard Hughes Medical Institute/ -- New York, N.Y. -- Science. 2012 Aug 17;337(6096):816-21. doi: 10.1126/science.1225829. Epub 2012 Jun 28.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/22745249" target="_blank"〉PubMed〈/a〉
    Keywords: Bacteriophages/*immunology ; Base Sequence ; *DNA Breaks, Double-Stranded ; *DNA Cleavage ; Deoxyribonucleases, Type II Site-Specific/chemistry/genetics/*metabolism ; *Inverted Repeat Sequences ; Molecular Sequence Data ; Nucleic Acid Conformation ; Plasmids/metabolism ; RNA/chemistry/*metabolism ; Streptococcus pyogenes/*enzymology/physiology
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 7
    Publication Date: 2014-02-08
    Description: Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4184034/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4184034/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Jinek, Martin -- Jiang, Fuguo -- Taylor, David W -- Sternberg, Samuel H -- Kaya, Emine -- Ma, Enbo -- Anders, Carolin -- Hauer, Michael -- Zhou, Kaihong -- Lin, Steven -- Kaplan, Matias -- Iavarone, Anthony T -- Charpentier, Emmanuelle -- Nogales, Eva -- Doudna, Jennifer A -- T32 GM066698/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- New York, N.Y. -- Science. 2014 Mar 14;343(6176):1247997. doi: 10.1126/science.1247997. Epub 2014 Feb 6.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/24505130" target="_blank"〉PubMed〈/a〉
    Keywords: Actinomyces/*enzymology ; Amino Acid Sequence ; Bacterial Proteins/*chemistry ; Caspase 9/*chemistry ; Crystallography, X-Ray ; DNA Cleavage ; Molecular Sequence Data ; Nucleic Acid Conformation ; Protein Structure, Secondary ; Protein Structure, Tertiary ; RNA/*chemistry ; Streptococcus pyogenes/*enzymology
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 8
    Publication Date: 2015-03-21
    Description: 〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394183/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394183/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Baltimore, David -- Berg, Paul -- Botchan, Michael -- Carroll, Dana -- Charo, R Alta -- Church, George -- Corn, Jacob E -- Daley, George Q -- Doudna, Jennifer A -- Fenner, Marsha -- Greely, Henry T -- Jinek, Martin -- Martin, G Steven -- Penhoet, Edward -- Puck, Jennifer -- Sternberg, Samuel H -- Weissman, Jonathan S -- Yamamoto, Keith R -- P50 HG005550/HG/NHGRI NIH HHS/ -- T32 GM066698/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- New York, N.Y. -- Science. 2015 Apr 3;348(6230):36-8. doi: 10.1126/science.aab1028. Epub 2015 Mar 19.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉California Institute of Technology, Mail Code 147-75, Pasadena, CA 91125, USA. ; Stanford University School of Medicine, 291 Campus Drive, Stanford, CA 94305, USA. ; University of California, Berkeley, 450 Li Ka Shing no. 3370, Berkeley, CA 94720-3370, USA. Innovative Genomics Initiative, University of California, Berkeley, 188 Li Ka Shing Center, Berkeley, CA 94720-3370, USA. ; Department of Biochemistry, University of Utah School of Medicine, 15 North Medical Drive East, Room 4100, Salt Lake City, UT 84112-5650, USA. ; Department of Medical History and Bioethics, School of Medicine and Public Health, University of Wisconsin Law School, 975 Bascom Mall, Madison, WI 53706, USA. ; Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. ; Innovative Genomics Initiative, University of California, Berkeley, 188 Li Ka Shing Center, Berkeley, CA 94720-3370, USA. ; Boston Children's Hospital, 300 Longwood Avenue, Karp Family Building, 7th Floor, Boston, MA 02115, USA. Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MD 20815, USA. ; Innovative Genomics Initiative, University of California, Berkeley, 188 Li Ka Shing Center, Berkeley, CA 94720-3370, USA. Departments of Molecular and Cell Biology and Chemistry, Howard Hughes Medical Institute, 731 Stanley Hall, MS 3220, University of California, Berkeley, Berkeley, CA 94720-3220, USA. doudna@berkeley.edu. ; Center for Law and the Biosciences, Crown Quadrangle 559 Nathan Abbott Way Stanford, CA 94305-8610, USA. ; Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. ; Department of Molecular and Cell Biology, College of Letters and Science, University of California, Berkeley, 210K Durant Hall, Berkeley, CA 94720-2920, USA. ; Alta Partners, One Embarcadero Center, 37th Floor, San Francisco, CA 94111, USA. ; Department of Pediatrics UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA 94143, USA. ; Department of Chemistry, 731 Stanley Hall, MS 3220, University of California, Berkeley, CA 94720-3220, USA. ; Innovative Genomics Initiative, University of California, Berkeley, 188 Li Ka Shing Center, Berkeley, CA 94720-3370, USA. Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, Byers Hall, 1700 4th Street, San Francisco, CA 94158-2330, USA. ; Innovative Genomics Initiative, University of California, Berkeley, 188 Li Ka Shing Center, Berkeley, CA 94720-3370, USA. UCSF School of Medicine, 600 16th Street, San Francisco, CA 94158, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25791083" target="_blank"〉PubMed〈/a〉
    Keywords: Biotechnology/ethics ; *Caspase 9 ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Transfer, Horizontal ; Genetic Engineering/*ethics ; Genetic Predisposition to Disease/*prevention & control ; Genome, Human/genetics ; Genomics ; *Germ Cells ; Humans ; Risk Management ; Targeted Gene Repair/*ethics
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 9
    Publication Date: 2014-01-28
    Description: In many bacteria and archaea, small RNAs derived from clustered regularly interspaced short palindromic repeats (CRISPRs) associate with CRISPR-associated (Cas) proteins to target foreign DNA for destruction. In Type I and III CRISPR/Cas systems, the Cas6 family of endoribonucleases generates functional CRISPR-derived RNAs by site-specific cleavage of repeat sequences in precursor transcripts. CRISPR repeats differ widely in both sequence and structure, with varying propensity to form hairpin folds immediately preceding the cleavage site. To investigate the evolution of distinct mechanisms for the recognition of diverse CRISPR repeats by Cas6 enzymes, we determined crystal structures of two Thermus thermophilus Cas6 enzymes both alone and bound to substrate and product RNAs. These structures show how the scaffold common to all Cas6 endonucleases has evolved two binding sites with distinct modes of RNA recognition: one specific for a hairpin fold and the other for a single-stranded 5'-terminal segment preceding the hairpin. These findings explain how divergent Cas6 enzymes have emerged to mediate highly selective pre-CRISPR-derived RNA processing across diverse CRISPR systems.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 10
    Publication Date: 2013-10-22
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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