RESUMEN

Precursor ribosomal RNA (pre-rRNA) processing is a key step in ribosome biosynthesis and involves numerous RNases. A HEPN (higher eukaryote and prokaryote nucleotide binding) nuclease Las1 and a polynucleotide kinase Grc3 assemble into a tetramerase responsible for rRNA maturation. Here, we report the structures of full-length Saccharomyces cerevisiae and Cyberlindnera jadinii Las1-Grc3 complexes, and C. jadinii Las1. The Las1-Grc3 structures show that the central coiled-coil domain of Las1 facilitates pre-rRNA binding and cleavage, while the Grc3 C-terminal loop motif directly binds to the HEPN active center of Las1 and regulates pre-rRNA cleavage. Structural comparison between Las1 and Las1-Grc3 complex exhibits that Grc3 binding induces conformational rearrangements of catalytic residues associated with HEPN nuclease activation. Biochemical assays identify that Las1 processes pre-rRNA at the two specific sites (C2 and C2'), which greatly facilitates rRNA maturation. Our structures and specific pre-rRNA cleavage findings provide crucial insights into the mechanism and pathway of pre-rRNA processing in ribosome biosynthesis.

Asunto(s)

Precursores del ARN , ARN Ribosómico , Ribosomas , Procesamiento Postranscripcional del ARN , Endonucleasas

RESUMEN

Toxin-antitoxin (TA) systems are widespread in bacteria and archaea plasmids and genomes to regulate DNA replication, gene transcription, or protein translation. Higher eukaryotic and prokaryotic nucleotide-binding (HEPN) and minimal nucleotidyltransferase (MNT) domains are prevalent in prokaryotic genomes and constitute TA pairs. However, three gene pairs (MTH304/305, 408/409, and 463/464) of Methanothermobacter thermautotropicus ΔH HEPN-MNT family have not been studied as TA systems. Among these candidates, our study characterizes the MTH463/MTH464 TA system. MTH463 expression inhibited Escherichia coli growth, whereas MTH464 did not and blocked MTH463 instead. Using site-directed MTH463 mutagenesis, we determined that amino acids R99G, H104A, and Y106A from the R[ɸX]4-6H motif are involved with MTH463 cell toxicity. Furthermore, we established that purified MTH463 could degrade MS2 phage RNA, whereas purified MTH464 neutralized MTH463 activity in vitro. Our results indicate that the endonuclease toxin MTH463 (encoding a HEPN domain) and its cognate antitoxin MTH464 (encoding the MNT domain) may act as a type II TA system in M. thermautotropicus ΔH. This study provides initial and essential information studying TA system functions, primarily archaea HEPN-MNT family.

Asunto(s)

Antitoxinas , Eucariontes , Nucleotidiltransferasas/metabolismo , Antitoxinas/genética , Células Procariotas , Methanobacteriaceae/genética , Proteínas Bacterianas/metabolismo

RESUMEN

Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide immunity against invading genetic elements such as bacteriophages and plasmids. In type III CRISPR systems, the recognition of target RNA leads to the synthesis of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins via their CRISPR-associated Rossmann fold (CARF) domains. Commonly, these are ribonucleases (RNases) that unspecifically degrade both invader and host RNA. To mitigate adverse effects on cell growth, ring nucleases can degrade extant cOAs to switch off ancillary nucleases. Here we show that the model organism Synechocystis sp. PCC 6803 harbors functional CARF-domain effector and ring nuclease proteins. We purified and characterized the two ancillary CARF-domain proteins from the III-D type CRISPR system of this cyanobacterium. The Csx1 homolog, SyCsx1, is a cyclic tetraadenylate(cA4)-dependent RNase with a strict specificity for cytosine nucleotides. The second CARF-domain protein with similarity to Csm6 effectors, SyCsm6, did not show RNase activity in vitro but was able to break down cOAs and attenuate SyCsx1 RNase activity. Our data suggest that the CRISPR systems in Synechocystis confer a multilayered cA4-mediated defense mechanism.

RESUMEN

HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) RNases are an emerging class of functionally diverse RNA processing and degradation enzymes. Members are defined by a small α-helical bundle encompassing a short consensus RNase motif. HEPN dimerization is a universal requirement for RNase activation as the conserved RNase motifs are precisely positioned at the dimer interface to form a composite catalytic center. While the core HEPN fold is conserved, the organization surrounding the HEPN dimer can support large structural deviations that contribute to their specialized functions. HEPN RNases are conserved throughout evolution and include bacterial HEPN RNases such as CRISPR-Cas and toxin-antitoxin associated nucleases, as well as eukaryotic HEPN RNases that adopt large multi-component machines. Here we summarize the canonical elements of the growing HEPN RNase family and identify molecular features that influence RNase function and regulation. We explore similarities and differences between members of the HEPN RNase family and describe the current mechanisms for HEPN RNase activation and inhibition.

Asunto(s)

Endorribonucleasas/metabolismo , Proteolisis , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Sistemas CRISPR-Cas , Dominio Catalítico , Endorribonucleasas/química , Endorribonucleasas/genética , Humanos , Conformación Proteica en Hélice alfa , Multimerización de Proteína , Estabilidad del ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Sistemas Toxina-Antitoxina

RESUMEN

The ribosome biogenesis factor Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif (RφXXXH) important for RNA cleavage. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las1 plays a fundamental role in processing pre-rRNA. Underscoring the significance of Las1 function in the cell, mutations in the human LAS1L (LAS1-like) gene have been associated with neurological dysfunction. Two juxtaposed HEPN nuclease motifs create Las1's composite nuclease active site, but the roles of the individual HEPN motif residues are poorly defined. Here using a combination of in vivo experiments in Saccharomyces cerevisiae and in vitro assays, we show that both HEPN nuclease motifs are required for Las1 nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we determined the consensus HEPN motif in the Las1 subfamily and uncovered its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN' chimeras, we defined the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las1 active site. We also established that conformational flexibility of the two HEPN domains is important for proper nuclease function. The results of our work reveal critical information about how dual HEPN domains come together to drive Las1-mediated RNA cleavage.

Asunto(s)

Endorribonucleasas/metabolismo , ARN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proliferación Celular , Secuencia de Consenso , Endorribonucleasas/química , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo

RESUMEN

The CRISPR-Cas13b system is a recently identified Class 2, RNA-targeting CRISPR-Cas system. The system has been repurposed to achieve robust mRNA knockdown and precise RNA-editing in mammalian cells. While the CRISPR-Cas13b system has become a powerful tool for nucleic acids manipulation, the mechanisms of the system are still not fully understood. Cas13b endonucleases from different bacterial species show poor overall sequence homologies, suggesting that structural (and probably functional) diversities may exist. It is therefore important to study CRISPR-Cas13b cases from different bacterial species. Here we report the expression, purification, and initial characterization of a Cas13b endonuclease that is associated with the 8th putative CRISPR locus from Porphyromonas gingivalis genome (Pgi8Cas13b). The full-length Pgi8Cas13b protein (1119 residues) was successfully expressed in E. Coli cells, and purified by affinity and ion-exchange chromatography methods. The purified protein is biologically active, being able to bind its cognate crRNA with high specificity and affinity. Preparation of biologically active Pgi8Cas13b protein provides the basis for further in vitro biochemical and biophysical studies of the Pgi8Cas13b CRISPR system.

Asunto(s)

Proteínas Asociadas a CRISPR , Porphyromonas gingivalis/genética , Proteínas Asociadas a CRISPR/biosíntesis , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endonucleasas/biosíntesis , Endonucleasas/química , Endonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Porphyromonas gingivalis/metabolismo , Proteínas Recombinantes

RESUMEN

The toxin-antitoxin (TA) systems have been attracting attention due to their role in regulating stress responses in prokaryotes and their biotechnological potential. Much recognition has been given to type II TA system of mesophiles, while thermophiles have received merely limited attention. Here, we are presenting the putative type II TA families encoded on the genomes of four Geobacillus strains. We employed the TA finder tool to mine for TA-coding genes and manually curated the results using protein domain analysis tools. We also used the NCBI BLAST, Operon Mapper, ProOpDB, and sequence alignment tools to reveal the geobacilli TA features. We identified 28 putative TA pairs, distributed over eight TA families. Among the identified TAs, 15 represent putative novel toxins and antitoxins, belonging to the MazEF, MNT-HEPN, ParDE, RelBE, and XRE-COG2856 TA families. We also identified a potentially new TA composite, AbrB-ParE. Furthermore, we are suggesting the Geobacillus acetyltransferase TA (GacTA) family, which potentially represents one of the unique TA families with a reverse gene order. Moreover, we are proposing a hypothesis on the xre-cog2856 gene expression regulation, which seems to involve the c-di-AMP. This study aims for highlighting the significance of studying TAs in Geobacillus and facilitating future experimental research.

Asunto(s)

Evolución Molecular , Regulación Bacteriana de la Expresión Génica/fisiología , Geobacillus , Familia de Multigenes/fisiología , Sistemas Toxina-Antitoxina/fisiología , Geobacillus/genética , Geobacillus/metabolismo

RESUMEN

Type III CRISPR effector complexes utilize a bound CRISPR RNA (crRNA) to detect the presence of RNA from invading mobile genetic elements in the cell. This RNA binding results in the activation of two enzymatic domains of the Cas10 subunit-the HD nuclease domain, which degrades DNA, and PALM/cyclase domain. The latter synthesizes cyclic oligoadenylate (cOA) molecules by polymerizing ATP, and cOA acts as a second messenger in the cell, switching on the antiviral response by activating host ribonucleases and other proteins. In this chapter, we focus on the methods required to study the biochemistry of this recently discovered cOA signaling pathway. We cover protein expression and purification, synthesis of cOA and its linear analogues, kinetic analysis of cOA synthesis and cOA-stimulated ribonuclease activity, and small molecule detection and identification with thin-layer chromatography and mass spectrometry. The methods described are based on our recent studies of the type III CRISPR system in Sulfolobus solfataricus, but are widely applicable to other type III systems.

Asunto(s)

Nucleótidos de Adenina/metabolismo , Proteínas Arqueales/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Oligorribonucleótidos/metabolismo , Sulfolobus solfataricus/metabolismo , Nucleótidos de Adenina/genética , Proteínas Arqueales/genética , Proteínas Asociadas a CRISPR/genética , Clonación Molecular/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Escherichia coli/genética , Cinética , Oligorribonucleótidos/genética , Sistemas de Mensajero Secundario , Transducción de Señal , Sulfolobus solfataricus/genética

RESUMEN

CRISPR-Cas systems provide prokaryotes with RNA-based adaptive immunity against viruses and plasmids. A unique feature of Type III CRISPR-Cas systems is that they selectively target transcriptionally-active invader DNA, and can cleave both the expressed RNA transcripts and source DNA. The Type III-A effector crRNP (CRISPR RNA-Cas protein complex), which contains Cas proteins Csm1-5, recognizes and degrades invader RNA and DNA in a crRNA-guided, manner. Interestingly, Type III-A systems also employ Csm6, an HEPN family ribonuclease that does not stably associate with the Type III-A effector crRNP, but nevertheless contributes to defense via mechanistic details that are still being determined. Here, we investigated the mechanism of action of Csm6 in Type III-A CRISPR-Cas systems from Lactococcus lactis, Staphylococcus epidermidis, and Streptococcus thermophilus expressed in Escherichia coli. We found that L. lactis and S. epidermidis Csm6 cleave RNA specifically after purines in vitro, similar to the activity reported for S. thermophilus Csm6. Moreover, L. lactis Csm6 functions as a divalent metal-independent, single strand-specific endoribonuclease that depends on the conserved HEPN domain. In vivo, we show that deletion of csm6 or expression of an RNase-defective form of Csm6 disrupts crRNA-dependent loss of plasmid DNA in all three systems expressed in E. coli. Mutations in the Csm1 palm domain, which are known to deactivate Csm6 ribonuclease activity, also prevent plasmid loss in the three systems. The results indicate that Csm6 ribonuclease activity rather than Csm1-mediated DNase activity effects anti-plasmid immunity by the three Type III-A systems investigated.

Asunto(s)

Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Inmunidad , Plásmidos/genética , Ribonucleasas/metabolismo , Secuencia de Bases , Endorribonucleasas/metabolismo , Inmunidad/efectos de los fármacos , Lactobacillus/efectos de los fármacos , Lactobacillus/genética , Metales/farmacología , Mutación/genética , Purinas/metabolismo , Staphylococcus epidermidis/genética

RESUMEN

Here we highlight the Grc3/Las1 complex, an essential RNA processing machine that is well conserved across eukaryotes and required for processing the pre-ribosomal RNA (pre-rRNA). Las1 is an endoribonuclease that cleaves the pre-rRNA while Grc3 is a polynucleotide kinase that phosphorylates the Las1-cleaved RNA product. Recently we showed that Grc3 and Las1 assemble into a higher-order complex composed of a dimer of Grc3/Las1 heterodimers that is required for nuclease and kinase activity. Unexpectedly, we found that the Grc3/Las1 complex draws numerous parallels with two other eukaryotic nucleases, Ire1 and RNase L. In this perspective we explore the similarities and differences between this family of nuclease integrated kinase super assemblies (NiKs) and their distinct roles in RNA cleavage.

Asunto(s)

Endorribonucleasas/metabolismo , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Procesamiento Postranscripcional del ARN , Animales , ADN Intergénico , Endorribonucleasas/química , Endorribonucleasas/genética , Regulación de la Expresión Génica , Humanos , Familia de Multigenes , Polinucleótido 5'-Hidroxil-Quinasa/química , Polinucleótido 5'-Hidroxil-Quinasa/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Especificidad por Sustrato

RESUMEN

Las1 is a recently discovered endoribonuclease that collaborates with Grc3-Rat1-Rai1 to process precursor ribosomal RNA (rRNA), yet its mechanism of action remains unknown. Disruption of the mammalian Las1 gene has been linked to congenital lethal motor neuron disease and X-linked intellectual disability disorders, thus highlighting the necessity to understand Las1 regulation and function. Here, we report that the essential Las1 endoribonuclease requires its binding partner, the polynucleotide kinase Grc3, for specific C2 cleavage. Our results establish that Grc3 drives Las1 endoribonuclease cleavage to its targeted C2 site both in vitro and in Saccharomyces cerevisiae. Moreover, we observed Las1-dependent activation of the Grc3 kinase activity exclusively toward single-stranded RNA. Together, Las1 and Grc3 assemble into a tetrameric complex that is required for competent rRNA processing. The tetrameric Grc3/Las1 cross talk draws unexpected parallels to endoribonucleases RNaseL and Ire1, and establishes Grc3/Las1 as a unique member of the RNaseL/Ire1 RNA splicing family. Together, our work provides mechanistic insight for the regulation of the Las1 endoribonuclease and identifies the tetrameric Grc3/Las1 complex as a unique example of a protein-guided programmable endoribonuclease.

Asunto(s)

Proteínas Nucleares/metabolismo , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Sitios de Unión , Escherichia coli/metabolismo , Proteínas Nucleares/genética , Fosforilación , Polinucleótido 5'-Hidroxil-Quinasa/genética , Dominios Proteicos , Multimerización de Proteína , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/análisis , Proteínas Recombinantes/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Técnicas del Sistema de Dos Híbridos

RESUMEN

C2c2, the effector of type VI CRISPR-Cas systems, has two RNase activities-one for cutting its RNA target and the other for processing the CRISPR RNA (crRNA). Here, we report the structures of Leptotrichia shahii C2c2 in its crRNA-free and crRNA-bound states. While C2c2 has a bilobed structure reminiscent of all other Class 2 effectors, it also exhibits different structural characteristics. It contains the REC lobe with a Helical-1 domain and the NUC lobe with two HEPN domains. The two RNase catalytic pockets responsible for cleaving pre-crRNA and target RNA are independently located on Helical-1 and HEPN domains, respectively. crRNA binding induces significant conformational changes that are likely to stabilize crRNA binding and facilitate target RNA recognition. These structures provide important insights into the molecular mechanism of dual RNase activities of C2c2 and establish a framework for its future engineering as a RNA editing tool.

Asunto(s)

Sistemas CRISPR-Cas , Leptotrichia/química , Leptotrichia/enzimología , Ribonucleasas/química , Secuencia de Aminoácidos , Dominio Catalítico , Leptotrichia/clasificación , Leptotrichia/metabolismo , Modelos Moleculares , Mutagénesis , Procesamiento Postranscripcional del ARN , ARN Bacteriano/química , ARN no Traducido/química , Alineación de Secuencia

RESUMEN

Prokaryotic CRISPR-Cas systems provide an RNA-guided mechanism for genome defense against mobile genetic elements such as viruses and plasmids. In type III-A CRISPR-Cas systems, the RNA-guided multisubunit Csm effector complex targets both single-stranded RNAs and double-stranded DNAs. In addition to the Csm complex, efficient anti-plasmid immunity mediated by type III-A systems also requires the CRISPR-associated protein Csm6. Here we report the crystal structure of Csm6 from Thermus thermophilus and show that the protein is a ssRNA-specific endoribonuclease. The structure reveals a dimeric architecture generated by interactions involving the N-terminal CARF and C-terminal HEPN domains. HEPN domain dimerization leads to the formation of a composite ribonuclease active site. Consistently, mutations of invariant active site residues impair catalytic activity in vitro. We further show that the ribonuclease activity of Csm6 is conserved across orthologs, suggesting that it plays an important functional role in CRISPR-Cas systems. The dimer interface of the CARF domains features a conserved electropositive pocket that may function as a ligand-binding site for allosteric control of ribonuclease activity. Altogether, our work suggests that Csm6 proteins provide an auxiliary RNA-targeting interference mechanism in type III-A CRISPR-Cas systems that operates in conjunction with the RNA- and DNA-targeting endonuclease activities of the Csm effector complex.

Asunto(s)

Proteínas Bacterianas/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endorribonucleasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Dominio Catalítico , Cristalografía por Rayos X , Dimerización , Endorribonucleasas/química , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido

RESUMEN

Prokaryotes are frequently exposed to potentially harmful invasive nucleic acids from phages, plasmids, and transposons. One method of defense is the CRISPR-Cas adaptive immune system. Diverse CRISPR-Cas systems form distinct ribonucleoprotein effector complexes that target and cleave invasive nucleic acids to provide immunity. The Type III-B Cmr effector complex has been found to target the RNA and DNA of the invader in the various bacterial and archaeal organisms where it has been characterized. Interestingly, the gene encoding the Csx1 protein is frequently located in close proximity to the Cmr1-6 genes in many genomes, implicating a role for Csx1 in Cmr function. However, evidence suggests that Csx1 is not a stably associated component of the Cmr effector complex, but is necessary for DNA silencing by the Cmr system in Sulfolobus islandicus. To investigate the function of the Csx1 protein, we characterized the activity of recombinant Pyrococcus furiosus Csx1 against various nucleic acid substrates. We show that Csx1 is a metal-independent, endoribonuclease that acts selectively on single-stranded RNA and cleaves specifically after adenosines. The RNA cleavage activity of Csx1 is dependent upon a conserved HEPN motif located within the C-terminal domain of the protein. This motif is also key for activity in other known ribonucleases. Collectively, the findings indicate that invader silencing by Type III-B CRISPR-Cas systems relies both on RNA and DNA nuclease activities from the Cmr effector complex as well as on the affiliated, trans-acting Csx1 endoribonuclease.

Asunto(s)

Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas , Endorribonucleasas/química , Pyrococcus furiosus/genética , ARN de Archaea/química , Adenosina/metabolismo , Secuencias de Aminoácidos , Proteínas Arqueales , Secuencia de Bases , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/inmunología , Endorribonucleasas/genética , Endorribonucleasas/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Pyrococcus furiosus/inmunología , ARN de Archaea/genética , ARN de Archaea/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sulfolobus/genética , Sulfolobus/inmunología

RESUMEN

Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements.

Asunto(s)

Bacterias , Proteínas Bacterianas , Sistemas CRISPR-Cas/fisiología , Evolución Molecular , ARN Bacteriano , Ribonucleasas , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Estructura Terciaria de Proteína , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Recombinación Genética/fisiología , Ribonucleasas/genética , Ribonucleasas/metabolismo

RESUMEN

Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance.

Asunto(s)

Endorribonucleasas/química , Endorribonucleasas/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura , Factores de Transcripción/química , Factores de Transcripción/ultraestructura , Difracción de Rayos X/métodos , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño