Samuel H Sternberg

University of California, Berkeley, Berkeley, CA, United States

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Publications (7)130.88 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: Type II CRISPR-Cas 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. Here, we report 2.6 and 2.2 Å resolution crystal structures of two major Cas9 enzymes 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.
    Science 02/2014; · 31.20 Impact Factor
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    ABSTRACT: 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.
    Nature 01/2014; · 38.60 Impact Factor
  • Biophysical Journal 01/2013; 104(2):198-. · 3.67 Impact Factor
  • Source
    Rachel E Haurwitz, Samuel H Sternberg, Jennifer A Doudna
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    ABSTRACT: CRISPR-Cas adaptive immune systems protect prokaryotes against foreign genetic elements. crRNAs derived from CRISPR loci base pair with complementary nucleic acids, leading to their destruction. In Pseudomonas aeruginosa, crRNA biogenesis requires the endoribonuclease Csy4, which binds and cleaves the repetitive sequence of the CRISPR transcript. Biochemical assays and three co-crystal structures of wild-type and mutant Csy4/RNA complexes reveal a substrate positioning and cleavage mechanism in which a histidine deprotonates the ribosyl 2'-hydroxyl pinned in place by a serine, leading to nucleophilic attack on the scissile phosphate. The active site catalytic dyad lacks a general acid to protonate the leaving group and positively charged residues to stabilize the transition state, explaining why the observed catalytic rate constant is ∼10(4)-fold slower than that of RNase A. We show that this RNA cleavage step is essential for assembly of the Csy protein-crRNA complex that facilitates target recognition. Considering that Csy4 recognizes a single cellular substrate and sequesters the cleavage product, evolutionary pressure has likely selected for substrate specificity and high-affinity crRNA interactions at the expense of rapid cleavage kinetics.
    The EMBO Journal 04/2012; 31(12):2824-32. · 9.82 Impact Factor
  • Source
    Samuel H Sternberg, Rachel E Haurwitz, Jennifer A Doudna
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    ABSTRACT: Bacteria and archaea possess adaptive immune systems that rely on small RNAs for defense against invasive genetic elements. CRISPR (clustered regularly interspaced short palindromic repeats) genomic loci are transcribed as long precursor RNAs, which must be enzymatically cleaved to generate mature CRISPR-derived RNAs (crRNAs) that serve as guides for foreign nucleic acid targeting and degradation. This processing occurs within the repetitive sequence and is catalyzed by a dedicated Cas6 family member in many CRISPR systems. In Pseudomonas aeruginosa, crRNA biogenesis requires the endoribonuclease Csy4 (Cas6f), which binds and cleaves at the 3' side of a stable RNA stem-loop structure encoded by the CRISPR repeat. We show here that Csy4 recognizes its RNA substrate with an ~50 pM equilibrium dissociation constant, making it one of the highest-affinity protein:RNA interactions of this size reported to date. Tight binding is mediated exclusively by interactions upstream of the scissile phosphate that allow Csy4 to remain bound to its product and thereby sequester the crRNA for downstream targeting. Substrate specificity is achieved by RNA major groove contacts that are highly sensitive to helical geometry, as well as a strict preference for guanosine adjacent to the scissile phosphate in the active site. Collectively, our data highlight diverse modes of substrate recognition employed by Csy4 to enable accurate selection of CRISPR transcripts while avoiding spurious, off-target RNA binding and cleavage.
    RNA 02/2012; 18(4):661-72. · 5.09 Impact Factor
  • Blake Wiedenheft, Samuel H Sternberg, Jennifer A Doudna
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    ABSTRACT: Clustered regularly interspaced short palindromic repeat (CRISPR) are essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. Similar to RNA interference (RNAi) pathways in eukaryotes, CRISPR-mediated immune systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids, including viruses and plasmids. However, the mechanism of RNA-based bacterial immunity is distinct from RNAi. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems.
    Nature 02/2012; 482(7385):331-8. · 38.60 Impact Factor
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    ABSTRACT: The specialized ribonuclease Dicer plays a central role in eukaryotic gene expression by producing small regulatory RNAs-microRNAs (miRNAs) and short interfering RNAs (siRNAs)-from larger double-stranded RNA (dsRNA) substrates. Although Dicer will cleave both imperfectly base-paired hairpin structures (pre-miRNAs) and perfect duplexes (pre-siRNAs) in vitro, it has not been clear whether these are mechanistically equivalent substrates and how dsRNA binding proteins such as trans-activation response (TAR) RNA binding protein (TRBP) influence substrate selection and RNA processing efficiency. We show here that human Dicer is much faster at processing a pre-miRNA substrate compared to a pre-siRNA substrate under both single and multiple turnover conditions. Maximal cleavage rates (V(max)) calculated by Michaelis-Menten analysis differed by more than 100-fold under multiple turnover conditions. TRBP was found to enhance dicing of both substrates to similar extents, and this stimulation required the two N-terminal dsRNA binding domains of TRBP. These results demonstrate that multiple factors influence dicing kinetics. While TRBP stimulates dicing by enhancing the stability of Dicer-substrate complexes, Dicer itself generates product RNAs at rates determined at least in part by the structural properties of the substrate.
    Journal of Molecular Biology 10/2010; 404(3):392-402. · 3.91 Impact Factor