Cascading into focus Yan Zhang and Erik J. Sontheimer Science 345, 1452 (2014); DOI: 10.1126/science.1260026
This copy is for your personal, non-commercial use only.
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here.
The following resources related to this article are available online at www.sciencemag.org (this information is current as of October 18, 2014 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/345/6203/1452.full.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/345/6203/1452.full.html#related This article cites 12 articles, 5 of which can be accessed free: http://www.sciencemag.org/content/345/6203/1452.full.html#ref-list-1 This article appears in the following subject collections: Biochemistry http://www.sciencemag.org/cgi/collection/biochem
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on October 18, 2014
Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here.
INSIGHTS | P E R S P E C T I V E S
Rotating target wheel
Recoil separator Q Mo/W/Sg trajectories
Q
Beam Beam/background trajectories
D
N2 (I) cryostat
Gas chromatography column RTC
Structures of a multisubunit protein-RNA complex reveal how the CRISPR system recognizes DNA targets By Yan Zhang and Erik J. Sontheimer
Tefon capillary 2⫻ 32 SiO2-covered detectors for α and β particles and fssion fragments Making Sg carbonyl. In Even et al.’s experiment, hot Sg atoms are produced in a nuclear reaction, transported through the GARIS magnetic separator, and slowed down in the recoil transfer chamber (RTC), where they react with CO. The resulting Sg(CO)6 products are transported to the thermochromatography apparatus for analysis.
metal (4, 5). Gas-phase chemical studies have been important tools for studying the chemical behavior of short-lived elements. The use of physical recoil separators that isolate the nuclear reaction products has allowed the synthesis of compounds, such as the metal carbonyls. The metal carbonyls of the group 6 elements—Cr(CO)6, Mo(CO)6, and W(CO)6—are well known. U(CO)6 is unstable above 30 K, in accord with predictions of relativistic quantum chemistry (6). However, Sg(CO)6 is predicted (6) to be stable because of relativistic effects that lead to stronger molecular bonding. Actinide carbonyls have been difficult both to synthesize and characterize. Laser ablation experiments produce U(CO)6 and Th(CO)6 only as transient species. The synthesis of transactinide carbonyls looked to be forbidding because of the harsh synthesis conditions required to create these elements. Even et al. use a novel separation of the recoiling reaction products to overcome these problems. Having synthesized a Sg carbonyl complex, they used thermochromatography to characterize it. The authors carried out the experiment at the GARIS recoil separator at the RIKEN accelerator facility in Japan. They first generated Mo, W, and Sg nuclei in nuclear reactions at the target portion of the separator (see the second figure). The recoiling nuclei were then separated from the incident projectile beam by the magnetic elements of the GARIS separator and entered a recoil transfer chamber. In that chamber, the Mo, W, and Sg nuclei were thermalized in a He/ CO mixture and reacted with the CO molecules to form carbonyls. These compounds were transported to the gas chromatography 1452
Cascading into focus
column, where a temperature gradient from 25°C to –120°C was maintained. The column consisted of 32 pairs of Si semiconductor detectors covered with SiO2 that recorded the decay of the radioactive nuclei. Mo and W formed volatile carbonyls that adsorbed on the column in temperature regions characteristic of their known enthalpies of adsorption. A volatile Sg species adsorbed on the column at a similar position. Monte Carlo simulations of the adsorption process allowed the scientists to deduce an adsorption enthalpy of 50 ± 4 kJ/mol for the Sg species, in good agreement with the theoretical predictions (7) and similar to the measured value for W(CO)6. Even et al. conclude that they have formed a volatile Sg carbonyl complex, probably Sg(CO)6. This represents the first synthesis of a new class of superheavy compounds— a finding that could lead to the synthesis of carbonyl complexes of elements 104 to 109. The implications of this development are widespread. For example, the chemistry of element 109, Mt, has not been studied. Using carbonyl complexes, it should now be possible to study Mt chemistry. Thermal dissociation experiments with the carbonyls should enable studies of the strengths of metal-carbon bonds in all the superheavy elements. ■ REFERENCES
1. 2. 3. 4. 5. 6.
J. Even et al., Science 345, 1491 (2014). M. Schädel, Radiochim. Acta 100, 579 (2012). R. Eichler et al., Nature 447, 72 (2007). R. Eichler et al., Radiochim. Acta 98, 133 (2010). A. Yakushev et al., Inorg. Chem. 53, 1624 (2014). C. S. Nash, B. E. Bursten, J. Am. Chem. Soc. 121, 10830 (1999). 7. V. Pershina, J. Anton, J. Chem. Phys. 138, 174301 (2013). 10.1126/science.1259349
A
n adaptive immune pathway in bacteria and archaea is specified by clustered, regularly interspaced, short palindromic repeat (CRISPR) sequences (1). Research into CRISPR RNAs (crRNAs) and the CRISPRassociated (Cas) proteins have revealed the ability of most of these systems to target foreign DNA molecules for destruction. On pages 1473 and 1479 of this issue, Jackson et al. (2) and Mulepati et al. (3), respectively, and Zhao et al. (4) describe high-resolution structures of the multiprotein assembly called CRISPR-associated complex for antiviral defense (“Cascade”), which drives CRISPR interference in many strains of Escherichia coli. The structures show how Cascade presents crRNA to its DNA target, and demonstrate that DNA recognition occurs through a configuration that, surprisingly, is not double-helical. Diverse flavors of CRISPR systems have been organized into three families (types I, II, and III) with additional subdivisions (5). All use crRNA precursors (pre-crRNAs) with multiple repeat sequences that are separated by “spacers.” These spacers match “protospacer” sequences that are usually present within phage genomes or plasmids. Pre-crRNAs are processed into individual crRNAs, each with a single spacer that guides the effector machinery to its DNA target. Type II systems require only a single protein, Cas9, for crRNA-guided DNA targeting. By contrast, crRNAs in type I and type III systems assemble into large multiprotein complexes. Cascade was first defined for the type I-E system from E. coli strain K12 (6) and includes one to six copies each of five distinct Cas proteins. Many of E. coli Cascade’s important interactions, mechanistic features (6–12), and structural properties (7–9, 12) have been described, including its seahorse-like shape. Double-stranded DNA (dsDNA) recognition by Cascade requires not only crRNA-DNA complementarity, but also a 3–base pair sciencemag.org SCIENCE
19 SEP TEMBER 2014 • VOL 345 ISSUE 6203
Published by AAAS
ILLUSTRATION: ADAPTED BY P. HUEY/SCIENCE
Mylar window
D
STRUCTURAL BIOLOGY
ILLUSTRATION: H. MCDONALD/SCIENCE
protospacer adjacent motif “seed,” which in E. coli lies (PAM) (6–11). Target recogniwithin the 5′-terminal eight Target DNA tion by Cascade activates a spacer nucleotides (10). Misseparate protein, Cas3, which matches in the seed are far Cas6e uses its nuclease domain to more deleterious to CRISPR 3’ destroy the target dsDNA (11). interference than are misHead crRNA Jackson et al. and Zhao et matches elsewhere (1). One al. report the structure of the unexpected feature of the E. Cas7.1 405-kilodalton E. coli Cascoli seed’s stringent complecade in its pre–target-bound mentarity requirement is that state. The complex contains 12 a single position within it—the Cas7.2 components (11 proteins, one sixth nucleotide—is excluded Cse2.1 RNA) and exposes most of the (10). The Cascade-DNA struc32-nucleotide crRNA spacer ture, with its unpaired conCas7.3 Belly (see the figure). The head of the figuration at precisely that Backbone seahorse-like complex includes position, explains why. In the Cas6e pre–crRNA-processaddition, the apo structures Cse2.2 Cas7.4 ing enzyme, tightly bound to suggest that all five of the disthe 3′-terminal stem-loop of played pentanucleotide blocks the crRNA. The “head” and (not just those that include Cas7.5 “tail” regions are connected seed residues) are preordered along the complex’s “backfor DNA pairing, which sugbone” by a helical arrangement gests that the seed’s disproCas7.6 of six Cas7 proteins (Cas7.1 to portionate importance is not 7.6), as well as a “belly” that due to a unique entropic adincludes two Cse2 proteins. In vantage. Instead, Jackson et Tail Cas5e the tail, the 5′-terminal crRNA al. suggest that the seed bases 5’ “handle” anchors the assembly are crucial to nucleate crRNAof the Cas5e, Cse1, and Cas7.6 DNA duplex formation beCse1 proteins. The Cas5e and Cas7 cause of their proximity to the subunits each resemble the PAM, the recognition of which shape of a right hand with fincould initiate local unwinding. ger, palm, and thumb-like doZhao et al. also point out that mains. The thumb of one Cas7 the seed region is relatively extends toward the fingers of Views of engagement. The Cascade multiprotein complex has a seahorse-like shape. Its free of obstruction by the Cse2 the preceding Cas7, stabilizing 11 protein subunits associate with crRNA. The cRNA then forms a ribbonlike duplex with dimer along the belly. Furthe Cas7 filament. Cas7.1 (at the target DNA. Six “kinked” nucleotides in the crRNA stick outward, allowing five regions of ther work will be required to head) and Cas7.6 (at the tail) contact with DNA. understand PAM recognition, cap the filament via distinct inas the complementary-strand teractions with Cas6e and Cas5e, respectively. Mulepati et al. report the structure of PAM nucleotides are disordered in Mulepati One of the most intriguing aspects of these E. coli Cascade bound to a single-stranded et al.’s structure and the other DNA strand structures is the apparent manner of crRNA DNA target. The overall architecture of is absent. presentation to target DNA. The thumbs of the DNA-bound complex is in superb Nonetheless, the three structures provide five Cas7 proteins (Cas7.2 to 7.6) project out agreement with the two apo structures of the first high-resolution views of Cascade, and fold over every sixth nucleotide of the Jackson et al. and Zhao et al., and further before and after engagement with its target crRNA, with concomitant introduction of reveals the configuration of the crRNADNA. Additional frames of this “movie” will a kink at each of those sites. Consequently, DNA duplex. Sure enough, the DNA is hopefully reveal how subsequent Cas3-catathe crRNA is displayed as five discrete penengaged in 5–base pair blocks, with every lyzed DNA degradation occurs. ■ tanucleotide segments, each poised for base sixth base unpaired. REFERENCES pairing with the target DNA. The bases of This underwound, ribbonlike arrange1. R. Barrangou, L. A. Marraffini, Mol. Cell 54, 234 (2014). the kinked nucleotides are flipped outward ment allows the target DNA recognition 2. R. N. Jackson, C. M. Lee, S. Taylor, Science 345, 1473 and apparently unavailable to participate in pathway to circumvent a potential topo(2014). 3. S. Mulepati, A. Héroux, S. Bailey, Science 345, 1479 (2014). DNA recognition. Functional tests demonlogical challenge. If the target DNA strand 4. H. Zhao et al., Nature 10.1038/nature13733 (2014). strate that DNA recognition is insensitive to were to be bound as a fully paired double 5. K. S. Makarova et al., Nat. Rev. Microbiol. 9, 467 (2011). mismatches at kinked nucleotides, whereas helix, it would either have to thread the gap 6. S. J. Brouns et al., Science 321, 960 (2008). 7. M. L. Hochstrasser et al., Proc. Natl. Acad. Sci. U.S.A. 111, mismatches elsewhere have much more dra(twice!) between the crRNA and Cascade, or 6618 (2014). matic effects on binding. The half-helicalthe complex would have to risk releasing a 8. M. M. Jore et al., Nat. Struct. Mol. Biol. 18, 529 (2011). turn segments in the crRNA suggest that the crRNA end to allow it to wrap around the 9. D. G. Sashital, B. Wiedenheft, J. A. Doudna, Mol. Cell 46, 606 (2012). DNA protospacer could be recognized in fiveDNA strand. Instead, each unpaired position 10. E. Semenova et al., Proc. Natl. Acad. Sci. U.S.A. 108, 10098 nucleotide blocks. enables the DNA to reset its trajectory for the (2011). next 5–base pair block without continuing 11. E. R. Westra et al., Mol. Cell 46, 595 (2012). 12. B. Wiedenheft et al., Nature 477, 486 (2011). around the other side of the crRNA. RNA Therapeutics Institute, Program in Molecular Medicine, DNA binding energetics is dominated by University of Massachusetts Medical School, Worcester, MA 01605, USA. E-mail: [email protected] 10.1126/science.1260026 a subset of crRNA positions known as the SCIENCE sciencemag.org
19 SEP TEMBER 2014 • VOL 345 ISSUE 6203
Published by AAAS
1453
This copy is for your personal, non-commercial use only.
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here.
The following resources related to this article are available online at www.sciencemag.org (this information is current as of October 18, 2014 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/345/6203/1452.full.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/345/6203/1452.full.html#related This article cites 12 articles, 5 of which can be accessed free: http://www.sciencemag.org/content/345/6203/1452.full.html#ref-list-1 This article appears in the following subject collections: Biochemistry http://www.sciencemag.org/cgi/collection/biochem
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on October 18, 2014
Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here.
INSIGHTS | P E R S P E C T I V E S
Rotating target wheel
Recoil separator Q Mo/W/Sg trajectories
Q
Beam Beam/background trajectories
D
N2 (I) cryostat
Gas chromatography column RTC
Structures of a multisubunit protein-RNA complex reveal how the CRISPR system recognizes DNA targets By Yan Zhang and Erik J. Sontheimer
Tefon capillary 2⫻ 32 SiO2-covered detectors for α and β particles and fssion fragments Making Sg carbonyl. In Even et al.’s experiment, hot Sg atoms are produced in a nuclear reaction, transported through the GARIS magnetic separator, and slowed down in the recoil transfer chamber (RTC), where they react with CO. The resulting Sg(CO)6 products are transported to the thermochromatography apparatus for analysis.
metal (4, 5). Gas-phase chemical studies have been important tools for studying the chemical behavior of short-lived elements. The use of physical recoil separators that isolate the nuclear reaction products has allowed the synthesis of compounds, such as the metal carbonyls. The metal carbonyls of the group 6 elements—Cr(CO)6, Mo(CO)6, and W(CO)6—are well known. U(CO)6 is unstable above 30 K, in accord with predictions of relativistic quantum chemistry (6). However, Sg(CO)6 is predicted (6) to be stable because of relativistic effects that lead to stronger molecular bonding. Actinide carbonyls have been difficult both to synthesize and characterize. Laser ablation experiments produce U(CO)6 and Th(CO)6 only as transient species. The synthesis of transactinide carbonyls looked to be forbidding because of the harsh synthesis conditions required to create these elements. Even et al. use a novel separation of the recoiling reaction products to overcome these problems. Having synthesized a Sg carbonyl complex, they used thermochromatography to characterize it. The authors carried out the experiment at the GARIS recoil separator at the RIKEN accelerator facility in Japan. They first generated Mo, W, and Sg nuclei in nuclear reactions at the target portion of the separator (see the second figure). The recoiling nuclei were then separated from the incident projectile beam by the magnetic elements of the GARIS separator and entered a recoil transfer chamber. In that chamber, the Mo, W, and Sg nuclei were thermalized in a He/ CO mixture and reacted with the CO molecules to form carbonyls. These compounds were transported to the gas chromatography 1452
Cascading into focus
column, where a temperature gradient from 25°C to –120°C was maintained. The column consisted of 32 pairs of Si semiconductor detectors covered with SiO2 that recorded the decay of the radioactive nuclei. Mo and W formed volatile carbonyls that adsorbed on the column in temperature regions characteristic of their known enthalpies of adsorption. A volatile Sg species adsorbed on the column at a similar position. Monte Carlo simulations of the adsorption process allowed the scientists to deduce an adsorption enthalpy of 50 ± 4 kJ/mol for the Sg species, in good agreement with the theoretical predictions (7) and similar to the measured value for W(CO)6. Even et al. conclude that they have formed a volatile Sg carbonyl complex, probably Sg(CO)6. This represents the first synthesis of a new class of superheavy compounds— a finding that could lead to the synthesis of carbonyl complexes of elements 104 to 109. The implications of this development are widespread. For example, the chemistry of element 109, Mt, has not been studied. Using carbonyl complexes, it should now be possible to study Mt chemistry. Thermal dissociation experiments with the carbonyls should enable studies of the strengths of metal-carbon bonds in all the superheavy elements. ■ REFERENCES
1. 2. 3. 4. 5. 6.
J. Even et al., Science 345, 1491 (2014). M. Schädel, Radiochim. Acta 100, 579 (2012). R. Eichler et al., Nature 447, 72 (2007). R. Eichler et al., Radiochim. Acta 98, 133 (2010). A. Yakushev et al., Inorg. Chem. 53, 1624 (2014). C. S. Nash, B. E. Bursten, J. Am. Chem. Soc. 121, 10830 (1999). 7. V. Pershina, J. Anton, J. Chem. Phys. 138, 174301 (2013). 10.1126/science.1259349
A
n adaptive immune pathway in bacteria and archaea is specified by clustered, regularly interspaced, short palindromic repeat (CRISPR) sequences (1). Research into CRISPR RNAs (crRNAs) and the CRISPRassociated (Cas) proteins have revealed the ability of most of these systems to target foreign DNA molecules for destruction. On pages 1473 and 1479 of this issue, Jackson et al. (2) and Mulepati et al. (3), respectively, and Zhao et al. (4) describe high-resolution structures of the multiprotein assembly called CRISPR-associated complex for antiviral defense (“Cascade”), which drives CRISPR interference in many strains of Escherichia coli. The structures show how Cascade presents crRNA to its DNA target, and demonstrate that DNA recognition occurs through a configuration that, surprisingly, is not double-helical. Diverse flavors of CRISPR systems have been organized into three families (types I, II, and III) with additional subdivisions (5). All use crRNA precursors (pre-crRNAs) with multiple repeat sequences that are separated by “spacers.” These spacers match “protospacer” sequences that are usually present within phage genomes or plasmids. Pre-crRNAs are processed into individual crRNAs, each with a single spacer that guides the effector machinery to its DNA target. Type II systems require only a single protein, Cas9, for crRNA-guided DNA targeting. By contrast, crRNAs in type I and type III systems assemble into large multiprotein complexes. Cascade was first defined for the type I-E system from E. coli strain K12 (6) and includes one to six copies each of five distinct Cas proteins. Many of E. coli Cascade’s important interactions, mechanistic features (6–12), and structural properties (7–9, 12) have been described, including its seahorse-like shape. Double-stranded DNA (dsDNA) recognition by Cascade requires not only crRNA-DNA complementarity, but also a 3–base pair sciencemag.org SCIENCE
19 SEP TEMBER 2014 • VOL 345 ISSUE 6203
Published by AAAS
ILLUSTRATION: ADAPTED BY P. HUEY/SCIENCE
Mylar window
D
STRUCTURAL BIOLOGY
ILLUSTRATION: H. MCDONALD/SCIENCE
protospacer adjacent motif “seed,” which in E. coli lies (PAM) (6–11). Target recogniwithin the 5′-terminal eight Target DNA tion by Cascade activates a spacer nucleotides (10). Misseparate protein, Cas3, which matches in the seed are far Cas6e uses its nuclease domain to more deleterious to CRISPR 3’ destroy the target dsDNA (11). interference than are misHead crRNA Jackson et al. and Zhao et matches elsewhere (1). One al. report the structure of the unexpected feature of the E. Cas7.1 405-kilodalton E. coli Cascoli seed’s stringent complecade in its pre–target-bound mentarity requirement is that state. The complex contains 12 a single position within it—the Cas7.2 components (11 proteins, one sixth nucleotide—is excluded Cse2.1 RNA) and exposes most of the (10). The Cascade-DNA struc32-nucleotide crRNA spacer ture, with its unpaired conCas7.3 Belly (see the figure). The head of the figuration at precisely that Backbone seahorse-like complex includes position, explains why. In the Cas6e pre–crRNA-processaddition, the apo structures Cse2.2 Cas7.4 ing enzyme, tightly bound to suggest that all five of the disthe 3′-terminal stem-loop of played pentanucleotide blocks the crRNA. The “head” and (not just those that include Cas7.5 “tail” regions are connected seed residues) are preordered along the complex’s “backfor DNA pairing, which sugbone” by a helical arrangement gests that the seed’s disproCas7.6 of six Cas7 proteins (Cas7.1 to portionate importance is not 7.6), as well as a “belly” that due to a unique entropic adincludes two Cse2 proteins. In vantage. Instead, Jackson et Tail Cas5e the tail, the 5′-terminal crRNA al. suggest that the seed bases 5’ “handle” anchors the assembly are crucial to nucleate crRNAof the Cas5e, Cse1, and Cas7.6 DNA duplex formation beCse1 proteins. The Cas5e and Cas7 cause of their proximity to the subunits each resemble the PAM, the recognition of which shape of a right hand with fincould initiate local unwinding. ger, palm, and thumb-like doZhao et al. also point out that mains. The thumb of one Cas7 the seed region is relatively extends toward the fingers of Views of engagement. The Cascade multiprotein complex has a seahorse-like shape. Its free of obstruction by the Cse2 the preceding Cas7, stabilizing 11 protein subunits associate with crRNA. The cRNA then forms a ribbonlike duplex with dimer along the belly. Furthe Cas7 filament. Cas7.1 (at the target DNA. Six “kinked” nucleotides in the crRNA stick outward, allowing five regions of ther work will be required to head) and Cas7.6 (at the tail) contact with DNA. understand PAM recognition, cap the filament via distinct inas the complementary-strand teractions with Cas6e and Cas5e, respectively. Mulepati et al. report the structure of PAM nucleotides are disordered in Mulepati One of the most intriguing aspects of these E. coli Cascade bound to a single-stranded et al.’s structure and the other DNA strand structures is the apparent manner of crRNA DNA target. The overall architecture of is absent. presentation to target DNA. The thumbs of the DNA-bound complex is in superb Nonetheless, the three structures provide five Cas7 proteins (Cas7.2 to 7.6) project out agreement with the two apo structures of the first high-resolution views of Cascade, and fold over every sixth nucleotide of the Jackson et al. and Zhao et al., and further before and after engagement with its target crRNA, with concomitant introduction of reveals the configuration of the crRNADNA. Additional frames of this “movie” will a kink at each of those sites. Consequently, DNA duplex. Sure enough, the DNA is hopefully reveal how subsequent Cas3-catathe crRNA is displayed as five discrete penengaged in 5–base pair blocks, with every lyzed DNA degradation occurs. ■ tanucleotide segments, each poised for base sixth base unpaired. REFERENCES pairing with the target DNA. The bases of This underwound, ribbonlike arrange1. R. Barrangou, L. A. Marraffini, Mol. Cell 54, 234 (2014). the kinked nucleotides are flipped outward ment allows the target DNA recognition 2. R. N. Jackson, C. M. Lee, S. Taylor, Science 345, 1473 and apparently unavailable to participate in pathway to circumvent a potential topo(2014). 3. S. Mulepati, A. Héroux, S. Bailey, Science 345, 1479 (2014). DNA recognition. Functional tests demonlogical challenge. If the target DNA strand 4. H. Zhao et al., Nature 10.1038/nature13733 (2014). strate that DNA recognition is insensitive to were to be bound as a fully paired double 5. K. S. Makarova et al., Nat. Rev. Microbiol. 9, 467 (2011). mismatches at kinked nucleotides, whereas helix, it would either have to thread the gap 6. S. J. Brouns et al., Science 321, 960 (2008). 7. M. L. Hochstrasser et al., Proc. Natl. Acad. Sci. U.S.A. 111, mismatches elsewhere have much more dra(twice!) between the crRNA and Cascade, or 6618 (2014). matic effects on binding. The half-helicalthe complex would have to risk releasing a 8. M. M. Jore et al., Nat. Struct. Mol. Biol. 18, 529 (2011). turn segments in the crRNA suggest that the crRNA end to allow it to wrap around the 9. D. G. Sashital, B. Wiedenheft, J. A. Doudna, Mol. Cell 46, 606 (2012). DNA protospacer could be recognized in fiveDNA strand. Instead, each unpaired position 10. E. Semenova et al., Proc. Natl. Acad. Sci. U.S.A. 108, 10098 nucleotide blocks. enables the DNA to reset its trajectory for the (2011). next 5–base pair block without continuing 11. E. R. Westra et al., Mol. Cell 46, 595 (2012). 12. B. Wiedenheft et al., Nature 477, 486 (2011). around the other side of the crRNA. RNA Therapeutics Institute, Program in Molecular Medicine, DNA binding energetics is dominated by University of Massachusetts Medical School, Worcester, MA 01605, USA. E-mail: [email protected] 10.1126/science.1260026 a subset of crRNA positions known as the SCIENCE sciencemag.org
19 SEP TEMBER 2014 • VOL 345 ISSUE 6203
Published by AAAS
1453
