RESUMO
The Cas9-crRNA complex of the Streptococcus thermophilus DGCC7710 CRISPR3-Cas system functions as an RNA-guided endonuclease with crRNA-directed target sequence recognition and protein-mediated DNA cleavage. We show here that an additional RNA molecule, tracrRNA (trans-activating CRISPR RNA), co-purifies with the Cas9 protein isolated from the heterologous E. coli strain carrying the S. thermophilus DGCC7710 CRISPR3-Cas system. We provide experimental evidence that tracrRNA is required for Cas9-mediated DNA interference both in vitro and in vivo. We show that Cas9 specifically promotes duplex formation between the precursor crRNA (pre-crRNA) transcript and tracrRNA, in vitro. Furthermore, the housekeeping RNase III contributes to primary pre-crRNA-tracrRNA duplex cleavage for mature crRNA biogenesis. RNase III, however, is not required in the processing of a short pre-crRNA transcribed from a minimal CRISPR array containing a single spacer. Finally, we show that an in vitro-assembled ternary Cas9-crRNA-tracrRNA complex cleaves DNA. This study further specifies the molecular basis for crRNA-based re-programming of Cas9 to specifically cleave any target DNA sequence for precise genome surgery. The processes for crRNA maturation and effector complex assembly established here will contribute to the further development of the Cas9 re-programmable system for genome editing applications.
Assuntos
Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , RNA Bacteriano/genética , Ribonuclease III/metabolismo , Streptococcus thermophilus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/genética , DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Ribonuclease III/química , Ribonuclease III/genética , Alinhamento de Sequência , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Streptococcus thermophilus/metabolismoRESUMO
The stressosome is a pseudo-icosahedral megadalton bacterial stress-sensing protein complex consisting of several copies of two STAS-domain proteins, RsbR and RsbS, and the kinase RsbT. Upon perception of environmental stress multiple copies of RsbT are released from the surface of the stressosome. Free RsbT activates downstream proteins to elicit a global cellular response, such as the activation of the general stress response in Gram-positive bacteria. The molecular events triggering RsbT release from the stressosome surface remain poorly understood. Here we present the map of Listeria innocua RsbR1/RsbS complex at resolutions of 3.45 Å for the STAS domain core in icosahedral symmetry and of 3.87 Å for the STAS domain and N-terminal sensors in D2 symmetry, respectively. The structure reveals a conformational change in the STAS domain linked to phosphorylation in RsbR. Docking studies indicate that allosteric RsbT binding to the conformationally flexible N-terminal sensor domain of RsbR affects the affinity of RsbS towards RsbT. Our results bring to focus the molecular events within the stressosome complex and further our understanding of this ubiquitous signaling hub.