RESUMEN
CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2-18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Humanos , Proteínas Bacterianas/metabolismo , Sistemas CRISPR-Cas/genética , ADN , ADN de Cadena Simple/genética , Edición Génica/métodos , Proteínas Asociadas a CRISPR , EndodesoxirribonucleasasRESUMEN
Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9-guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family.
Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas CRISPR-Cas/fisiología , ADN de Cadena Simple/metabolismo , Endonucleasas/metabolismo , Streptococcus pyogenes/enzimología , Proteínas Bacterianas/genética , ADN de Cadena Simple/genética , Endonucleasas/genética , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Streptococcus pyogenes/genéticaRESUMEN
CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.
Asunto(s)
Sistemas CRISPR-Cas , Endonucleasas/metabolismo , Edición Génica , Línea Celular Tumoral , Endonucleasas/genética , Células Hep G2 , Humanos , Estructura Molecular , Ingeniería de ProteínasRESUMEN
Bacteria and archaea acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA into clustered regularly interspaced short palindromic repeats (CRISPRs). These repetitive loci maintain a genetic record of all prior encounters with foreign transgressors. CRISPRs are transcribed and the long primary transcript is processed into a library of short CRISPR-derived RNAs (crRNAs) that contain a unique sequence complementary to a foreign nucleic-acid challenger. In Escherichia coli, crRNAs are incorporated into a multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defence), which is required for protection against bacteriophages. Here we use cryo-electron microscopy to determine the subnanometre structures of Cascade before and after binding to a target sequence. These structures reveal a sea-horse-shaped architecture in which the crRNA is displayed along a helical arrangement of protein subunits that protect the crRNA from degradation while maintaining its availability for base pairing. Cascade engages invading nucleic acids through high-affinity base-pairing interactions near the 5' end of the crRNA. Base pairing extends along the crRNA, resulting in a series of short helical segments that trigger a concerted conformational change. This conformational rearrangement may serve as a signal that recruits a trans-acting nuclease (Cas3) for destruction of invading nucleic-acid sequences.
Asunto(s)
Escherichia coli K12/genética , Escherichia coli K12/inmunología , Proteínas de Escherichia coli/ultraestructura , Sustancias Macromoleculares/química , Sustancias Macromoleculares/ultraestructura , ARN Bacteriano/inmunología , ARN Bacteriano/ultraestructura , Emparejamiento Base , Microscopía por Crioelectrón , Escherichia coli K12/química , Escherichia coli K12/virología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/inmunología , Secuencias Invertidas Repetidas/genética , Secuencias Invertidas Repetidas/inmunología , Sustancias Macromoleculares/metabolismo , Modelos Biológicos , Modelos Moleculares , Conformación Proteica , ARN Bacteriano/genéticaRESUMEN
RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA-protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5' ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.
Asunto(s)
Proteínas Bacterianas/metabolismo , Endorribonucleasas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuencias Invertidas Repetidas/genética , MicroARNs/análisis , Proteómica/métodos , Proteínas de Unión al ARN/análisis , Secuencia de Bases , Western Blotting , Proteínas Asociadas a CRISPR , Células HeLa , Humanos , Imidazoles , Espectrometría de Masas , MicroARNs/genética , MicroARNs/aislamiento & purificación , Datos de Secuencia Molecular , Ingeniería de Proteínas/métodos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Análisis de Secuencia de ARNRESUMEN
The 3' untranslated region (3'UTR) of hepatitis C virus (HCV) messenger RNA stimulates viral translation by an undetermined mechanism. We identified a high affinity interaction, conserved among different HCV genotypes, between the HCV 3'UTR and the host ribosome. The 3'UTR interacts with 40S ribosomal subunit proteins residing primarily in a localized region on the 40S solvent-accessible surface near the messenger RNA entry and exit sites. This region partially overlaps with the site where the HCV internal ribosome entry site was found to bind, with the internal ribosome entry site-40S subunit interaction being dominant. Despite its ability to bind to 40S subunits independently, the HCV 3'UTR only stimulates translation in cis, without affecting the first round translation rate. These observations support a model in which the HCV 3'UTR retains ribosome complexes during translation termination to facilitate efficient initiation of subsequent rounds of translation.
Asunto(s)
Regiones no Traducidas 3' , Regulación Viral de la Expresión Génica , Hepacivirus/genética , Biosíntesis de Proteínas , Regiones no Traducidas 5' , Línea Celular Tumoral , Factor 3 de Iniciación Eucariótica/metabolismo , Humanos , ARN Viral/química , ARN Viral/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/virologíaRESUMEN
During RNA interference and related gene regulatory pathways, the endonuclease Dicer cleaves precursor RNA molecules to produce microRNAs (miRNAs) and short interfering RNAs (siRNAs). Human cells encode a single Dicer enzyme that can associate with two different double-stranded RNA (dsRNA)-binding proteins, protein activator of PKR (PACT) and trans-activation response RNA-binding protein (TRBP). However, the functional redundancy or differentiation of PACT and TRBP in miRNA and siRNA biogenesis is not well understood. Using a reconstituted system, we show here that PACT and TRBP have distinct effects on Dicer-mediated dsRNA processing. In particular, we found that PACT in complex with Dicer inhibits the processing of pre-siRNA substrates when compared with Dicer and a Dicer-TRBP complex. In addition, PACT and TRBP show non-redundant effects on the production of different-sized miRNAs (isomiRs), which in turn alter target-binding specificities. Experiments using chimeric versions of PACT and TRBP suggest that the two N-terminal RNA-binding domains of each protein confer the observed differences in dsRNA substrate recognition and processing behavior of Dicer-dsRNA-binding protein complexes. These results support the conclusion that in humans, Dicer-associated dsRNA-binding proteins are important regulatory factors that contribute both substrate and cleavage specificity during miRNA and siRNA production.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismo , Proteínas Argonautas/metabolismo , Humanos , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/fisiologíaRESUMEN
Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated (cas) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a "seed sequence" at the 5' end of the CRISPR RNA spacer. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli, termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems.
Asunto(s)
Secuencia de Bases , Escherichia coli , Sustancias Macromoleculares/metabolismo , Pseudomonas aeruginosa , ARN/genética , ARN/metabolismo , Inmunidad Adaptativa/genética , Inmunidad Adaptativa/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/inmunología , Sustancias Macromoleculares/química , Modelos Moleculares , Datos de Secuencia Molecular , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunologíaRESUMEN
A hallmark of RNA interference is the production of short double-stranded RNA (dsRNA) molecules 21-28 nucleotides in length by the specialized RNase III protein Dicer. Dicer enzymes uniquely generate RNA products of specific lengths by mechanisms that have not been fully elucidated. Here we show that the PAZ domain responsible for dsRNA end recognition confers this measuring ability through both its structural position and RNA-binding specificity. Point mutations define the dsRNA-binding surface and reveal a protein loop important for cleavage of substrates containing perfect or imperfect base pairing. On the basis of these results, we reengineered Dicer with a U1A RNA-binding domain in place of the PAZ domain to create an enzyme with altered end-recognition specificity and RNA product length. These results explain how Dicer functions as a molecular ruler and provide a structural basis for modifying its activity in cells.
Asunto(s)
Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , ARN Bicatenario/metabolismo , Ribonucleasa III/química , Ribonucleasa III/metabolismo , Animales , Secuencia de Bases , Giardia lamblia/enzimología , Giardia lamblia/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Puntual , Proteínas Protozoarias/genética , ARN Bicatenario/genética , Ribonucleasa III/genética , Electricidad EstáticaRESUMEN
Ribozymes enhance chemical reaction rates using many of the same catalytic strategies as protein enzymes. In the hepatitis delta virus (HDV) ribozyme, site-specific self-cleavage of the viral RNA phosphodiester backbone requires both divalent cations and a cytidine nucleotide. General acid-base catalysis, substrate destabilization and global and local conformational changes have all been proposed to contribute to the ribozyme catalytic mechanism. Here we report ten crystal structures of the HDV ribozyme in its pre-cleaved state, showing that cytidine is positioned to activate the 2'-OH nucleophile in the precursor structure. This observation supports its proposed role as a general base in the reaction mechanism. Comparison of crystal structures of the ribozyme in the pre- and post-cleavage states reveals a significant conformational change in the RNA after cleavage and that a catalytically critical divalent metal ion from the active site is ejected. The HDV ribozyme has remarkable chemical similarity to protein ribonucleases and to zymogens for which conformational dynamics are integral to biological activity. This finding implies that RNA structural rearrangements control the reactivity of ribozymes and ribonucleoprotein enzymes.
Asunto(s)
Virus de la Hepatitis Delta/enzimología , Virus de la Hepatitis Delta/genética , Conformación de Ácido Nucleico , ARN Catalítico/química , ARN Catalítico/metabolismo , Sitios de Unión , Catálisis , Cationes Bivalentes/metabolismo , Cristalografía por Rayos X , Citidina/genética , Citidina/metabolismo , Concentración de Iones de Hidrógeno , Metales/metabolismo , Modelos Moleculares , Mutación/genética , ARN Catalítico/genéticaRESUMEN
Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , División del ADN , ADN/química , Endonucleasas/química , Streptococcus pyogenes/enzimología , Dominio Catalítico , Cristalografía por Rayos X , Endonucleasas/ultraestructura , Ingeniería Genética , Genoma , Conformación de Ácido Nucleico , Conformación Proteica , ARN/química , ARN Guía de KinetoplastidaRESUMEN
Bacterial adaptive immunity uses CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR transcripts for foreign DNA degradation. In type II CRISPR-Cas systems, activation of Cas9 endonuclease for DNA recognition upon guide RNA binding occurs by an unknown mechanism. Crystal structures of Cas9 bound to single-guide RNA reveal a conformation distinct from both the apo and DNA-bound states, in which the 10-nucleotide RNA "seed" sequence required for initial DNA interrogation is preordered in an A-form conformation. This segment of the guide RNA is essential for Cas9 to form a DNA recognition-competent structure that is poised to engage double-stranded DNA target sequences. We construe this as convergent evolution of a "seed" mechanism reminiscent of that used by Argonaute proteins during RNA interference in eukaryotes.
Asunto(s)
Proteínas Argonautas/química , Proteínas Bacterianas/química , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , División del ADN , Endonucleasas/química , ARN Guía de Kinetoplastida/química , Streptococcus pyogenes/enzimología , Proteínas Bacterianas/genética , Secuencia de Bases , Proteína 9 Asociada a CRISPR , Cristalografía por Rayos X , ADN/química , Endonucleasas/genética , Activación Enzimática , Evolución Molecular , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , Interferencia de ARNRESUMEN
HIV replication requires nuclear export of unspliced and singly spliced viral transcripts. Although a unique RNA structure has been proposed for the Rev-response element (RRE) responsible for viral mRNA export, how it recruits multiple HIV Rev proteins to form an export complex has been unclear. We show here that initial binding of Rev to the RRE triggers RNA tertiary structural changes, enabling further Rev binding and the rapid formation of a viral export complex. Analysis of the Rev-RRE assembly pathway using SHAPE-Seq and small-angle X-ray scattering (SAXS) reveals two major steps of Rev-RRE complex formation, beginning with rapid Rev binding to a pre-organized region presenting multiple Rev binding sites. This step induces long-range remodeling of the RNA to expose a cryptic Rev binding site, enabling rapid assembly of additional Rev proteins into the RNA export complex. This kinetic pathway may help maintain the balance between viral replication and maturation.DOI: http://dx.doi.org/10.7554/eLife.03656.001.
Asunto(s)
Núcleo Celular/metabolismo , VIH-1/química , ARN Guía de Kinetoplastida/química , ARN Viral/química , Elementos de Respuesta , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/química , Transporte Activo de Núcleo Celular/genética , Sitios de Unión , Núcleo Celular/virología , Células Eucariotas/virología , VIH-1/metabolismo , Humanos , Datos de Secuencia Molecular , Pliegue del ARN , Empalme del ARN , Transporte de ARN , ARN Guía de Kinetoplastida/metabolismo , ARN Viral/metabolismo , Dispersión del Ángulo Pequeño , Termodinámica , Replicación Viral , Difracción de Rayos X , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
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.
Asunto(s)
Actinomyces/enzimología , Proteínas Bacterianas/química , Endonucleasas/química , ARN/química , Streptococcus pyogenes/enzimología , Secuencia de Aminoácidos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cristalografía por Rayos X , División del ADN , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The conserved ribonuclease Dicer generates microRNAs and short-interfering RNAs that guide gene silencing in eukaryotes. The specific contributions of human Dicer's structural domains to RNA product length and substrate preference are incompletely understood, due in part to the difficulties of Dicer purification. Here, we show that active forms of human Dicer can be assembled from recombinant polypeptides expressed in bacteria. Using this system, we find that three distinct modes of RNA recognition give rise to Dicer's fidelity and product length specificity. The first involves anchoring one end of a double-stranded RNA helix within the PAZ domain, which can assemble in trans with Dicer's catalytic domains to reconstitute an accurate but non-substrate-selective dicing activity. The second entails nonspecific RNA binding by the double-stranded RNA binding domain, an interaction that is essential for substrate recruitment in the absence of the PAZ domain. The third mode of recognition involves hairpin RNA loop recognition by the helicase domain, which ensures efficient processing of specific substrates. These results reveal distinct interactions of each Dicer domain with different RNA structural features and provide a facile system for investigating the molecular mechanisms of human microRNA biogenesis.
Asunto(s)
ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Bacterias/genética , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA(1)B(2)C(6)D(1)E(1)) and a 61-nucleotide CRISPR RNA (crRNA) with 5'-hydroxyl and 2',3'-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.
Asunto(s)
ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/virología , Ribonucleoproteínas/química , Secuencia de Bases , Sitios de Unión , Escherichia coli/inmunología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Bacteriano/fisiología , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/fisiología , Relación Estructura-Actividad , ARN Pequeño no TraducidoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Procesamiento Postranscripcional del ARN , ARN Bacteriano/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Sustitución de Aminoácidos , Emparejamiento Base , Secuencia de Bases , Proteínas Asociadas a CRISPR , Cristalización , Cristalografía por Rayos X , Genes Bacterianos , Enlace de Hidrógeno , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , Estructura Terciaria de Proteína , ARN Bacteriano/química , ARN Bacteriano/genética , Electricidad EstáticaRESUMEN
Acquired immunity in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into clustered regularly interspaced short palindromic repeats (CRISPRs). This nucleic acid-based immune system is mediated by a variable cassette of up to 45 protein families that represent distinct immune system subtypes. CRISPR-associated gene 1 (cas1) encodes the only universally conserved protein component of CRISPR immune systems, yet its function is unknown. Here we show that the Cas1 protein is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments of approximately 80 base pairs in length. The 2.2 A crystal structure of the Cas1 protein reveals a distinct fold and a conserved divalent metal ion-binding site. Mutation of metal ion-binding residues, chelation of metal ions, or metal-ion substitution inhibits Cas1-catalyzed DNA degradation. These results provide a foundation for understanding how Cas1 contributes to CRISPR function, perhaps as part of the machinery for processing foreign nucleic acids.
Asunto(s)
Desoxirribonucleasas/química , Genoma , Proteínas/clasificación , Proteínas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Secuencia de Aminoácidos , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Desoxirribonucleasas/metabolismo , Dimerización , Genoma Bacteriano , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Células Procariotas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/aislamiento & purificaciónRESUMEN
Cellular internal ribosome entry sites (IRESs) are untranslated segments of mRNA transcripts thought to initiate protein synthesis in response to environmental stresses that prevent canonical 5' cap-dependent translation. Although numerous cellular mRNAs are proposed to have IRESs, none has a demonstrated physiological function or molecular mechanism. Here we show that seven yeast genes required for invasive growth, a developmental pathway induced by nutrient limitation, contain potent IRESs that require the initiation factor eIF4G for cap-independent translation. In contrast to the RNA structure-based activity of viral IRESs, we show that an unstructured A-rich element mediates internal initiation via recruitment of the poly(A) binding protein (Pab1) to the 5' untranslated region (UTR) of invasive growth messages. A 5'UTR mutation that impairs IRES activity compromises invasive growth, which indicates that cap-independent translation is required for physiological adaptation to stress.
Asunto(s)
Regiones no Traducidas 5' , Iniciación de la Cadena Peptídica Traduccional , ARN de Hongos/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/genética , Regiones no Traducidas 5'/genética , Regiones no Traducidas 5'/metabolismo , Adaptación Fisiológica , Factor 4G Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Genes Fúngicos , Glucosa/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Poli A/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Biosíntesis de Proteínas , Caperuzas de ARN/metabolismo , ARN de Hongos/química , ARN de Hongos/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/biosíntesis , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The specialized ribonuclease Dicer initiates RNA interference by cleaving double-stranded RNA (dsRNA) substrates into small fragments about 25 nucleotides in length. In the crystal structure of an intact Dicer enzyme, the PAZ domain, a module that binds the end of dsRNA, is separated from the two catalytic ribonuclease III (RNase III) domains by a flat, positively charged surface. The 65 angstrom distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA. Thus, Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end.