Scott Bailey

Crystal structure of a CRISPR RNA–guided surveillance complex bound to a ssDNA target

A foreign-DNA–destroying machine Bacteria have an adaptive immune system, called CRISPR, that identifies invading viruses through their DNA or RNA sequences and cuts them up (see the Perspective by Zhang and Sontheimer). Jackson et al. and Mulepati et al. have determined the structure of the large protein complex, called Cascade, that targets the invading nucleic acids and does the cutting. The seahorse-shaped structure reveals how the 11 subcomponents of Cascade assemble into the final protein complex. The structure also shows how Cascade presents the short CRISPR-derived RNAs so that they can bind and target foreign DNA. Science, this issue p. 1473 and p. 1479; see also p. 1452 The structure of the Cascade complex reveals how the bacterial CRISPR immune system targets foreign DNA. [Also see Perspective by Zhang and Sontheimer] In prokaryotes, RNA derived from type I and type III CRISPR loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. In Escherichia coli, this 405-kilodalton complex is called Cascade. We report the crystal structure of Cascade bound to a single-stranded DNA (ssDNA) target at a resolution of 3.03 angstroms. The structure reveals that the CRISPR RNA and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. This noncanonical structure is facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid and is stabilized by the highly interlocked organization of protein subunits. These studies provide insight into both the assembly and the activity of this complex and suggest a mechanism to enforce fidelity of target binding.

The Structure of T. aquaticus DNA Polymerase III Is Distinct from Eukaryotic Replicative DNA Polymerases.

The crystal structure of Thermus aquaticus DNA polymerase III alpha subunit reveals that the structure of the catalytic domain of the eubacterial replicative polymerase is unrelated to that of the eukaryotic replicative polymerase but rather belongs to the Polbeta-like nucleotidyltransferase superfamily. A model of the polymerase complexed with both DNA and beta-sliding clamp interacting with a reoriented binding domain and internal beta binding site was constructed that is consistent with existing biochemical data. Within the crystal, two C-terminal domains are interacting through a surface that is larger than many dimer interfaces. Since replicative polymerases of eubacteria and eukaryotes/archaea are not homologous, the nature of the replicative polymerase in the last common ancestor is unknown. Although other possibilities have been proposed, the plausibility of a ribozyme DNA polymerase should be considered.

In Vitro Reconstitution of an Escherichia coli RNA-guided Immune System Reveals Unidirectional, ATP-dependent Degradation of DNA Target

Background: The CRISPR-Cas immune system protects E. coli against invasive DNA. Results: We have reconstituted this immune system in vitro using recombinant proteins. Conclusion: Degradation of invasive DNA is tightly regulated and unidirectional. Significance: Reconstitution provides an invaluable tool for understanding the CRISPR-Cas immune system. Many prokaryotes utilize small RNA transcribed from clustered, regularly interspaced, short palindromic repeats (CRISPRs) to protect themselves from foreign genetic elements, such as phage and plasmids. In Escherichia coli, this small RNA is packaged into a surveillance complex (Cascade) that uses the RNA sequence to direct binding to invasive DNA. Once bound, Cascade recruits the Cas3 nuclease-helicase, which then proceeds to progressively degrade the invading DNA. Here, using individually purified Cascade and Cas3 from E. coli, we reconstitute CRISPR-mediated plasmid degradation in vitro. Analysis of this reconstituted assay suggests that Cascade recruits Cas3 to a single-stranded region of the DNA target exposed by Cascade binding. Cas3 then nicks the exposed DNA. Recruitment and nicking is stimulated by the presence, but not hydrolysis, of ATP. Following nicking and powered by ATP hydrolysis, the concerted actions of the helicase and nuclease domains of Cas3 proceed to unwind and degrade the entire DNA target in a unidirectional manner.

Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3).

RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.

The Structure of a Transcribing T7 RNA Polymerase in Transition from Initiation to Elongation

Structural studies of the T7 bacteriophage DNA-dependent RNA polymerase (T7 RNAP) have shown that the conformation of the amino-terminal domain changes substantially between the initiation and elongation phases of transcription, but how this transition is achieved remains unclear. We report crystal structures of T7 RNAP bound to promoter DNA containing either a 7- or an 8-nucleotide (nt) RNA transcript that illuminate intermediate states along the transition pathway. The amino-terminal domain comprises the C-helix subdomain and the promoter binding domain (PBD), which consists of two segments separated by subdomain H. The structures of the intermediate complex reveal that the PBD and the bound promoter rotate by ∼45° upon synthesis of an 8-nt RNA transcript. This allows the promoter contacts to be maintained while the active site is expanded to accommodate a growing heteroduplex. The C-helix subdomain moves modestly toward its elongation conformation, whereas subdomain H remains in its initiation- rather than its elongation-phase location, more than 70 angstroms away.

Structural basis for promiscuous PAM recognition in type I–E Cascade from E. coli

Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5′-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process.

Structure of the RAG1 Nonamer Binding Domain with DNA Reveals a Dimer that Mediates DNA Synapsis

The products of recombination-activating genes RAG1 and RAG2 mediate the assembly of antigen receptor genes during lymphocyte development in a process known as V(D)J recombination. Lack of structural information for the RAG proteins has hindered mechanistic studies of this reaction. We report here the crystal structure of an essential DNA binding domain of the RAG1 catalytic core bound to its nonamer DNA recognition motif. The RAG1 nonamer binding domain (NBD) forms a tightly interwoven dimer that binds and synapses two nonamer elements, with each NBD making contact with both DNA molecules. Biochemical and biophysical experiments confirm that the two nonamers are in close proximity in the RAG1/2–DNA synaptic complex and demonstrate the functional importance of the protein-DNA contacts revealed in the structure. These findings reveal a previously unsuspected function for the NBD in DNA synapsis and have implications for the regulation of DNA binding and cleavage by RAG1 and RAG2.

RNA-activated DNA cleavage by the Type III-B CRISPR–Cas effector complex

The CRISPR (clustered regularly interspaced short palindromic repeat) system is an RNA-guided immune system that protects prokaryotes from invading genetic elements. This system represents an inheritable and adaptable immune system that is mediated by multisubunit effector complexes. In the Type III-B system, the Cmr effector complex has been found to cleave ssRNA in vitro. However, in vivo, it has been implicated in transcription-dependent DNA targeting. We show here that the Cmr complex from Thermotoga maritima can cleave an ssRNA target that is complementary to the CRISPR RNA. We also show that binding of a complementary ssRNA target activates an ssDNA-specific nuclease activity in the histidine-aspartate (HD) domain of the Cmr2 subunit of the complex. These data suggest a mechanism for transcription-coupled DNA targeting by the Cmr complex and provide a unifying mechanism for all Type III systems.

Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease

Background: The Cas9 RNA-guided endonuclease has been adapted for genome manipulation and regulation. Results: We have characterized target recognition and cleavage by Streptococcus thermophilus LMG18311 Cas9. Conclusion: The two nuclease domains of Cas9 select their cleavage sites by different mechanisms. Significance: These findings contribute to the molecular basis of Cas9-mediated DNA cleavage. Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat–CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.

Metalloprotease NleC Suppresses Host NF-κB/Inflammatory Responses by Cleaving p65 and Interfering with the p65/RPS3 Interaction

Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p651–38 fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p651–38 fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a ‘specifier’ subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.