RESUMO
DNA can form many structures beyond the canonical Watson-Crick double helix. It is now clear that noncanonical structures are present in genomic DNA and have biological functions. G-rich G-quadruplexes and C-rich i-motifs are the most well-characterized noncanonical DNA motifs that have been detected in vivo with either proscribed or postulated biological roles. Because of their independent sequence requirements, these structures have largely been considered distinct types of quadruplexes. Here, we describe the crystal structure of the DNA oligonucleotide, d(CCAGGCTGCAA), that self-associates to form a quadruplex structure containing two central antiparallel G-tetrads and six i-motif C-C+ base pairs. Solution studies suggest a robust structural motif capable of assembling as a tetramer of individual strands or as a dimer when composed of tandem repeats. This hybrid structure highlights the growing structural diversity of DNA and suggests that biological systems may harbor many functionally important non-duplex structures.
Assuntos
Pareamento de Bases/fisiologia , DNA/química , Quadruplex G , Motivos de Nucleotídeos/fisiologia , Bário/química , Bário/farmacologia , Pareamento de Bases/efeitos dos fármacos , Cristalografia por Raios X , Estabilidade de Medicamentos , Quadruplex G/efeitos dos fármacos , Ligação de Hidrogênio/efeitos dos fármacos , Modelos Moleculares , Conformação de Ácido Nucleico/efeitos dos fármacos , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Motivos de Nucleotídeos/efeitos dos fármacos , Oligonucleotídeos/químicaRESUMO
Noncanonical DNA structures that retain programmability and structural predictability are increasingly being used in DNA nanotechnology applications, in which they offer versatility beyond traditional Watson-Crick interactions. The d(CGA) triplet repeat motif is structurally dynamic and can transition between parallel-stranded homo-base paired duplex and antiparallel unimolecular hairpin in a pH-dependent manner. Here, we evaluate the thermodynamic stability and nuclease sensitivity of oligonucleotides composed of the d(CGA) motif and several structurally related sequence variants. These results show that the structural transition resulting from decreasing the pH is accompanied by both a significant energetic stabilization and decreased nuclease sensitivity as unimolecular hairpin structures are converted to parallel-stranded homo-base paired duplexes. Furthermore, the stability of the parallel-stranded duplex form can be altered by changing the 5'-nucleobase of the d(CGA) triplet and the frequency and position of the altered triplets within long stretches of d(CGA) triplets. This work offers insight into the stability and versatility of the d(CGA) triplet repeat motif and provides constraints for using this pH-adaptive structural motif for creating DNA-based nanomaterials.
Assuntos
DNA , Oligonucleotídeos , Pareamento de Bases , DNA/genética , Concentração de Íons de Hidrogênio , Conformação de Ácido NucleicoRESUMO
Guanosine-5'-hydroxamic acid (3) forms hydrogels when mixed with guanosine (1) and KCl. The 5'-hydroxamic acid (HA) unit is pH-responsive and also chelates Fe3+ . When gels are prepared under basic conditions, the 5'-HA groups are deprotonated and the anionic hydrogel binds cationic thiazole orange (TO), signaled by enhanced fluorescence. The HA nucleoside 3, when immobilized in the G-quartet gel, acts as a supramolecular siderophore to form red complexes with Fe3+ . We patterned the hydrogel's surface with FeCl3 , by hand and by using a 3D printer. Patterns form instantly, are visible by eye, and can be erased using vitaminâ C. This hydrogel, combining self-assembled G-quartet and siderophore-Fe3+ motifs, is strong, can be molded into different shapes, and is stable on the bench or under salt water.
RESUMO
DNA can adopt many structures beyond the Watson-Crick duplex. However, the bounds of DNA structural diversity and how these structures might regulate biological processes is only beginning to be understood. Here, we describe the 1.05 Å resolution crystal structure of a DNA oligonucleotide that self-associates to form a non-G-quadruplex fold-back structure. Distinct from previously described fold-back quadruplexes, two-fold-back dimers interact through noncanonical and Watson-Crick interactions to form a tetrameric assembly. These interactions include a hexad base pairing arrangement from two C-G-G base triples. The assembly is dependent on divalent cations, and the interface between the dimeric units creates a cavity in which a cation resides. This structure provides new sequence and structural contexts for the formation of fold-back quadruplexes, further highlighting the potential biological importance of this type of noncanonical DNA structure. This structure may also serve as the basis for designing new types of DNA nanoarchitectures or cation sensors based on the strong divalent cation dependence.
Assuntos
DNA/química , Oligodesoxirribonucleotídeos/química , Pareamento de Bases , Cristalografia por Raios X , DNA/genética , Magnésio/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/genéticaRESUMO
The mitochondrial tyrosyl-tRNA synthetases (mtTyrRSs) of Pezizomycotina fungi, a subphylum that includes many pathogenic species, are bifunctional proteins that both charge mitochondrial tRNA(Tyr) and act as splicing cofactors for autocatalytic group I introns. Previous studies showed that one of these proteins, Neurospora crassa CYT-18, binds group I introns by using both its N-terminal catalytic and C-terminal anticodon binding domains and that the catalytic domain uses a newly evolved group I intron binding surface that includes an N-terminal extension and two small insertions (insertions 1 and 2) with distinctive features not found in non-splicing mtTyrRSs. To explore how this RNA binding surface diverged to accommodate different group I introns in other Pezizomycotina fungi, we determined x-ray crystal structures of C-terminally truncated Aspergillus nidulans and Coccidioides posadasii mtTyrRSs. Comparisons with previous N. crassa CYT-18 structures and a structural model of the Aspergillus fumigatus mtTyrRS showed that the overall topology of the group I intron binding surface is conserved but with variations in key intron binding regions, particularly the Pezizomycotina-specific insertions. These insertions, which arose by expansion of flexible termini or internal loops, show greater variation in structure and amino acids potentially involved in group I intron binding than do neighboring protein core regions, which also function in intron binding but may be more constrained to preserve mtTyrRS activity. Our results suggest a structural basis for the intron specificity of different Pezizomycotina mtTyrRSs, highlight flexible terminal and loop regions as major sites for enzyme diversification, and identify targets for therapeutic intervention by disrupting an essential RNA-protein interaction in pathogenic fungi.
Assuntos
Aspergillus nidulans/enzimologia , Coccidioides/enzimologia , Íntrons/genética , Mitocôndrias/enzimologia , Splicing de RNA/genética , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/metabolismo , Sequência de Aminoácidos , Aspergillus nidulans/genética , Aspergillus nidulans/crescimento & desenvolvimento , Domínio Catalítico , Coccidioides/genética , Coccidioides/crescimento & desenvolvimento , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Neurospora crassa/enzimologia , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Tirosina-tRNA Ligase/genéticaRESUMO
DNA is now one of the most widely used molecules for programmed self-assembly of discrete nanostructures. One of the long-standing goals of the DNA nanotechnology field has been the assembly of periodic, macroscopic 3D DNA crystals for controlled positioning of guest molecules to be used in a variety of applications. With continuing successes in assembling DNA crystals, there is an enhanced need to tailor macroscopic crystal properties-including morphology-to enable their integration into more complex systems. Here we describe the ability to alter and control crystal habits of a 3D DNA crystal formed by self-assembly of a DNA 13-mer. The introduction of "poison" oligonucleotides that specifically disrupt critical noncanonical base-pairing interactions in the crystal lattice leads to predictably modified crystal habits. We demonstrate that the poison oligomers can act as habit modifiers both during the initial crystallization and during growth of shell layers on a crystal macroseed.
Assuntos
DNA/síntese química , Cristalização , DNA/química , Tamanho da PartículaRESUMO
OBJECTIVE: Understanding the mechanisms regulating normal and pathological angiogenesis is of great scientific and clinical interest. In this report, we show that mutations in 2 different aminoacyl-transfer RNA synthetases, threonyl tRNA synthetase (tars(y58)) or isoleucyl tRNA synthetase (iars(y68)), lead to similar increased branching angiogenesis in developing zebrafish. APPROACH AND RESULTS: The unfolded protein response pathway is activated by aminoacyl-transfer RNA synthetase deficiencies, and we show that unfolded protein response genes atf4, atf6, and xbp1, as well as the key proangiogenic ligand vascular endothelial growth factor (vegfaa), are all upregulated in tars(y58) and iars(y68) mutants. Finally, we show that the protein kinase RNA-like endoplasmic reticulum kinase-activating transcription factor 4 arm of the unfolded protein response pathway is necessary for both the elevated vegfaa levels and increased angiogenesis observed in tars(y58) mutants. CONCLUSIONS: Our results suggest that endoplasmic reticulum stress acts as a proangiogenic signal via unfolded protein response pathway-dependent upregulation of vegfaa.
Assuntos
Isoleucina-tRNA Ligase/deficiência , Neovascularização Fisiológica , Treonina-tRNA Ligase/deficiência , Resposta a Proteínas não Dobradas , Proteínas de Peixe-Zebra/deficiência , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Isoleucina-tRNA Ligase/genética , Mutação , Fenótipo , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Treonina-tRNA Ligase/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína 1 de Ligação a X-Box , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
DNA has proved to be an excellent material for nanoscale construction because complementary DNA duplexes are programmable and structurally predictable. However, in the absence of Watson-Crick pairings, DNA can be structurally more diverse. Here, we describe the crystal structures of d(ACTCGGATGAT) and the brominated derivative, d(AC(Br)UCGGA(Br)UGAT). These oligonucleotides form parallel-stranded duplexes with a crystallographically equivalent strand, resulting in the first examples of DNA crystal structures that contains four different symmetric homo base pairs. Two of the parallel-stranded duplexes are coaxially stacked in opposite directions and locked together to form a tetraplex through intercalation of the 5'-most A-A base pairs between adjacent G-G pairs in the partner duplex. The intercalation region is a new type of DNA tertiary structural motif with similarities to the i-motif. (1)H-(1)H nuclear magnetic resonance and native gel electrophoresis confirmed the formation of a parallel-stranded duplex in solution. Finally, we modified specific nucleotide positions and added d(GAY) motifs to oligonucleotides and were readily able to obtain similar crystals. This suggests that this parallel-stranded DNA structure may be useful in the rational design of DNA crystals and nanostructures.
Assuntos
DNA/química , Substâncias Intercalantes/química , Conformação de Ácido Nucleico , Halogênios/química , Espectroscopia de Ressonância MagnéticaRESUMO
Three-dimensional (3D) DNA crystals have been envisioned as a powerful tool for the positional control of biological and non-biological arrays on the nanoscale. However, most DNA crystals contain short duplex regions that can result in low thermal stability. Additionally, because DNA is a polyanion, DNA crystals often require high cation concentrations to maintain their integrity. Here, we demonstrate that a DNA alkylating mustard, bis(2-chloroethyl)amine, can form interstrand crosslinks within a model 3D DNA crystal. The crosslinking procedure did not alter crystal X-ray diffraction properties, but it did significantly improve the overall stability of the crystals under a variety of conditions. Crosslinked crystals showed enhanced stability at elevated temperature and were stable at Mg(2+) concentrations as low as 1â mm. Remarkably, the crosslinked crystals showed significant resistance to DNaseâ I treatment, while also having improved longevity in tissue culture mediums. Characterization of the crosslinked species suggest that there are multiple crosslinking sites, but that the most prevalent interstrand crosslink involves an unpaired 3'-terminal guanosine residue. The improved stability of these DNA crystals suggests that simple treatment with alkylating reagents might be sufficient to stabilize crystals and other DNA constructs for improved functionality in biological and non-biological applications.
Assuntos
Reagentes de Ligações Cruzadas/química , DNA/química , Desoxirribonuclease I/metabolismo , Conformação de Ácido Nucleico , Difração de Raios XRESUMO
DNA can adopt many other structures beyond the canonical B-form double helix. These alternative DNA structures have become increasingly significant as new biological roles are found for them. Additionally, there has been a growing interest in using non-canonical base pairs to provide structural diversity for designing DNA architectures for nanotechnology applications. We recently described the crystal structure of d(ACTCGGATGAT), which forms a tetraplex through parallel-stranded homo-base pairs and nucleobase intercalation. The homoduplex region contains a d(YGAâ YGA) motif observed in crystal and solution structures. Here, we examine the structural implications of the homopyrimidine base pair within this motif. We determined crystal structures of two variants that differ from the original structure in the homopyrimidine base pairs and number of d(YGA) motifs. Our results show that the intercalation-locked tetraplex motif is predictable in these different sequence contexts and that substituting Câ C base pairs for Tâ T base pairs introduces asymmetry to the homoduplex. These results have important implications for utilizing d(YGA) motifs in DNA crystal design and could provide a basis for understanding how local structures could be associated with repeat expansions.
Assuntos
Oligonucleotídeos/química , Pirimidinas/química , Pareamento de Bases , Cristalografia por Raios X , Citosina/química , Substâncias Intercalantes/química , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Oligonucleotídeos/síntese químicaRESUMO
We have determined the 1.50 Å crystal structure of the DNA decamer, d(CCA(CNV)KGCGTGG) ((CNV)K, 3-cyanovinylcarbazole), which forms a G-quadruplex structure in the presence of Ba(2+). The structure contains several unique features including a bulged nucleotide and the first crystal structure observation of a C-tetrad. The structure reveals that water molecules mediate contacts between the divalent cations and the C-tetrad, allowing Ba(2+) ions to occupy adjacent steps in the central ion channel. One ordered Mg(2+) facilitates 3'-3' stacking of two quadruplexes in the asymmetric unit, while the bulged nucleotide mediates crystal contacts. Despite the high diffraction limit, the first four nucleotides including the (CNV)K nucleoside are disordered though they are still involved in crystal packing. This work suggests that the bulky hydrophobic groups may locally influence the formation of non-Watson-Crick structures from otherwise complementary sequences. These observations lead to the intriguing possibility that certain types of DNA damage may act as modulators of G-quadruplex formation.
Assuntos
Bário/química , DNA/química , Quadruplex G , Água/química , Cátions Bivalentes , Cristalografia por Raios X , Magnésio , Modelos MolecularesRESUMO
DNA has proved to be a remarkable molecule for the construction of sophisticated two-dimensional and three-dimensional architectures because of its programmability and structural predictability provided by complementary Watson-Crick base pairing. DNA oligonucleotides can, however, exhibit a great deal of local structural diversity. DNA conformation is strongly linked to both environmental conditions and the nucleobase identities inherent in the oligonucleotide sequence, but the exact relationship between sequence and local structure is not completely understood. This study examines how a single-nucleotide addition to a class of self-assembling DNA 13-mers leads to a significantly different overall structure under identical crystallization conditions. The DNA 13-mers self-assemble in the presence of Mg(2+) through a combination of Watson-Crick and noncanonical base-pairing interactions. The crystal structures described here show that all of the predicted Watson-Crick base pairs are present, with the major difference being a significant rearrangement of noncanonical base pairs. This includes the formation of a sheared A-G base pair, a junction of strands formed from base-triple interactions, and tertiary interactions that generate structural features similar to tandem sheared G-A base pairs. The adoption of this alternate noncanonical structure is dependent in part on the sequence in the Watson-Crick duplex region. These results provide important new insights into the sequence-structure relationship of short DNA oligonucleotides and demonstrate a unique interplay between Watson-Crick and noncanonical base pairs that is responsible for crystallization fate.
Assuntos
DNA/química , Magnésio/química , Oligonucleotídeos/química , Pareamento de Bases , Sequência de Bases , Cátions Bivalentes , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , TermodinâmicaRESUMO
DNA is a widely used biopolymer for the construction of nanometer-scale objects due to its programmability and structural predictability. One long-standing goal of the DNA nanotechnology field has been the construction of three-dimensional DNA crystals. We previously determined the X-ray crystal structure of a DNA 13-mer that forms a continuously hydrogen bonded three-dimensional lattice through Watson-Crick and non-canonical base pairs. Our current study sets out to understand how the sequence of the Watson-Crick duplex region influences crystallization of this 13-mer. We screened all possible self-complementary sequences in the hexameric duplex region and found 21 oligonucleotides that crystallized. Sequence analysis showed that one specific Watson-Crick pair influenced the crystallization propensity and the speed of crystal self-assembly. We determined X-ray crystal structures for 13 of these oligonucleotides and found sequence-specific structural changes that suggests that this base pair may serve as a structural anchor during crystal assembly. Finally, we explored the crystal self-assembly and nucleation process. Solution studies indicated that these oligonucleotides do not form base pairs in the absence of cations, but that the addition of divalent cations leads to rapid self-assembly to higher molecular weight complexes. We further demonstrate that crystals grown from mixtures of two different oligonucleotide sequences contain both oligonucleotides. These results suggest that crystal self-assembly is nucleated by the formation of the Watson-Crick duplexes initiated by a simple chemical trigger. This study provides new insight into the role of sequence for the assembly of periodic DNA structures.
Assuntos
DNA/química , Cristalografia por Raios X , Conformação de Ácido Nucleico , Oligonucleotídeos/químicaRESUMO
The mitochondrial tyrosyl tRNA synthetase from Neurospora crassa (CYT-18 protein) is a bifunctional group I intron splicing cofactor. CYT-18 is capable of splicing multiple group I introns from a wide variety of sources by stabilizing the catalytically active intron structures. CYT-18 and mt TyrRSs from related fungal species have evolved to assist in group I intron splicing in part by the accumulation of three N-terminal domain insertions. Biochemical and structural analysis indicate that the N-terminal insertions serve primarily to create a structure-stabilizing scaffold for critical tertiary interactions between the two major RNA domains of group I introns. Previous studies concluded that the primarily α-helical N-terminal insertion, H0, contributes to protein stability and is necessary for splicing the N. crassa ND1 intron but is dispensable for splicing the N. crassa mitochondrial LSU intron. Here, we show that CYT-18 with a complete H0 deletion retains residual ND1 intron splicing activity and that addition of the missing N-terminus in trans is capable of restoring a significant portion of its splicing activity. The development of this peptide complementation assay has allowed us to explore important characteristics of the CYT-18/group I intron interaction including the stoichiometry of H0 in intron splicing and the importance of specific H0 residues. Evaluation of truncated H0 peptides in this assay and a re-examination of the CYT-18 crystal structure suggest a previously unknown structural role of the first five N-terminal residues of CYT-18. These residues interact directly with another splicing insertion, making H0 a central structural element responsible for connecting all three N-terminal splicing insertions.
Assuntos
Íntrons/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Splicing de RNA , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Estabilidade Enzimática , Modelos Moleculares , Dados de Sequência Molecular , Neurospora crassa/enzimologia , Conformação Proteica , Splicing de RNA/efeitos dos fármacos , Sais/farmacologia , TemperaturaRESUMO
Here we demonstrate that protein enzymes captured in the solvent channels of three-dimensional DNA crystals are catalytically active. Using RNase A as a model enzyme system, we show that crystals infused with enzyme can cleave a dinucleotide substrate with similar kinetic restrictions as other immobilized enzyme systems. This new vehicle for immobilized enzymes, created entirely from biomolecules, opens possibilities for developing modular solid-state catalysts that could be both biocompatible and biodegradable.
Assuntos
Biocatálise , DNA/química , Cristalização , Inibidores Enzimáticos/farmacologia , Enzimas Imobilizadas/química , Cinética , Nucleotídeos/química , Ribonuclease Pancreático/antagonistas & inibidores , Ribonuclease Pancreático/químicaRESUMO
The 'RNA world' hypothesis holds that during evolution the structural and enzymatic functions initially served by RNA were assumed by proteins, leading to the latter's domination of biological catalysis. This progression can still be seen in modern biology, where ribozymes, such as the ribosome and RNase P, have evolved into protein-dependent RNA catalysts ('RNPzymes'). Similarly, group I introns use RNA-catalysed splicing reactions, but many function as RNPzymes bound to proteins that stabilize their catalytically active RNA structure. One such protein, the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (TyrRS; CYT-18), is bifunctional and both aminoacylates mitochondrial tRNA(Tyr) and promotes the splicing of mitochondrial group I introns. Here we determine a 4.5-A co-crystal structure of the Twort orf142-I2 group I intron ribozyme bound to splicing-active, carboxy-terminally truncated CYT-18. The structure shows that the group I intron binds across the two subunits of the homodimeric protein with a newly evolved RNA-binding surface distinct from that which binds tRNA(Tyr). This RNA binding surface provides an extended scaffold for the phosphodiester backbone of the conserved catalytic core of the intron RNA, allowing the protein to promote the splicing of a wide variety of group I introns. The group I intron-binding surface includes three small insertions and additional structural adaptations relative to non-splicing bacterial TyrRSs, indicating a multistep adaptation for splicing function. The co-crystal structure provides insight into how CYT-18 promotes group I intron splicing, how it evolved to have this function, and how proteins could have incrementally replaced RNA structures during the transition from an RNA world to an RNP world.
Assuntos
Íntrons/genética , Neurospora crassa/enzimologia , Splicing de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Ligação Proteica , RNA/genética , RNA/metabolismo , RNA Catalítico/química , RNA Catalítico/genética , RNA Catalítico/metabolismo , Fagos de Staphylococcus/enzimologia , Fagos de Staphylococcus/genéticaRESUMO
BACKGROUND: The genera Aspergillus and Penicillium include some of the most beneficial as well as the most harmful fungal species such as the penicillin-producer Penicillium chrysogenum and the human pathogen Aspergillus fumigatus, respectively. Their mitochondrial genomic sequences may hold vital clues into the mechanisms of their evolution, population genetics, and biology, yet only a handful of these genomes have been fully sequenced and annotated. RESULTS: Here we report the complete sequence and annotation of the mitochondrial genomes of six Aspergillus and three Penicillium species: A. fumigatus, A. clavatus, A. oryzae, A. flavus, Neosartorya fischeri (A. fischerianus), A. terreus, P. chrysogenum, P. marneffei, and Talaromyces stipitatus (P. stipitatum). The accompanying comparative analysis of these and related publicly available mitochondrial genomes reveals wide variation in size (25-36 Kb) among these closely related fungi. The sources of genome expansion include group I introns and accessory genes encoding putative homing endonucleases, DNA and RNA polymerases (presumed to be of plasmid origin) and hypothetical proteins. The two smallest sequenced genomes (A. terreus and P. chrysogenum) do not contain introns in protein-coding genes, whereas the largest genome (T. stipitatus), contains a total of eleven introns. All of the sequenced genomes have a group I intron in the large ribosomal subunit RNA gene, suggesting that this intron is fixed in these species. Subsequent analysis of several A. fumigatus strains showed low intraspecies variation. This study also includes a phylogenetic analysis based on 14 concatenated core mitochondrial proteins. The phylogenetic tree has a different topology from published multilocus trees, highlighting the challenges still facing the Aspergillus systematics. CONCLUSIONS: The study expands the genomic resources available to fungal biologists by providing mitochondrial genomes with consistent annotations for future genetic, evolutionary and population studies. Despite the conservation of the core genes, the mitochondrial genomes of Aspergillus and Penicillium species examined here exhibit significant amount of interspecies variation. Most of this variation can be attributed to accessory genes and mobile introns, presumably acquired by horizontal gene transfer of mitochondrial plasmids and intron homing.
Assuntos
Aspergillus/genética , Genes Fúngicos/genética , Tamanho do Genoma/genética , Genoma Mitocondrial/genética , Íntrons/genética , Penicillium/genética , Análise de Sequência , Sequência de Bases , Evolução Molecular , Genes Mitocondriais/genética , Variação Genética/genética , Anotação de Sequência Molecular , Mutagênese Insercional/genética , Filogenia , Plasmídeos/genéticaRESUMO
Three-dimensional (3D) DNA crystals have been envisioned as programmable biomaterial scaffolds for creating ordered arrays of biological and nonbiological molecules. Despite having excellent programmable properties, the linearity of the Watson-Crick B-form duplex imposes limitations on 3D crystal design. Predictable noncanonical base pairing motifs have the potential to serve as junctions to connect linear DNA segments into complex 3D lattices. Here, we designed crystals based on a template structure with parallel-stranded noncanonical base pairs. Depending on pH, the structures we determined contained all but one or two of the designed secondary structure interactions. Surprisingly, a conformational change of the designed Watson-Crick duplex region resulted in crystal packing differences between the predicted and observed structures. However, the designed noncanonical motif was virtually identical to the template when crystals were grown at pH 5.5, highlighting the motif's predictability. At pH 7.0 we observed a structurally similar variation on this motif that contains a previously unobserved C-Gâ¢G-C quadruple base pair. We demonstrate that these two variants can interconvert in crystallo in response to pH perturbations. This study spotlights several important considerations in DNA crystal design, describes the first 3D DNA lattice composed of A-DNA helical sheets, and reveals a noncanonical DNA motif that has adaptive features that may be useful for designing dynamic crystals or biomaterial assemblies.
Assuntos
DNA/química , Pareamento de Bases , Sequência de Bases , Cristalização , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido NucleicoRESUMO
DNA can adopt noncanonical structures that have important biological functions while also providing structural diversity for applications in nanotechnology. Here, the crystal structures of two oligonucleotides composed of d(CGA) triplet repeats in the parallel-stranded duplex form are described. The structure determination of four unique d(CGA)-based parallel-stranded duplexes across two crystal structures has allowed the structural parameters of d(CGA) triplets in the parallel-stranded duplex form to be characterized and established. These results show that d(CGA) units are highly uniform, but that each strand in the duplex is structurally unique and has a distinct role in accommodating structural asymmetries induced by the C-CH+ base pair.
Assuntos
DNA , Oligonucleotídeos , Pareamento de Bases , DNA/química , Conformação de Ácido Nucleico , Oligonucleotídeos/químicaRESUMO
The mitochondrial tyrosyl-tRNA synthetases (mt TyrRSs) of Pezizomycotina fungi are bifunctional proteins that aminoacylate mitochondrial tRNA(Tyr) and are structure-stabilizing splicing cofactors for group I introns. Studies with the Neurospora crassa synthetase (CYT-18 protein) showed that splicing activity is dependent upon Pezizomycotina-specific structural adaptations that form a distinct group I intron-binding site in the N-terminal catalytic domain. Although CYT-18's C-terminal domain also binds group I introns, it has been intractable to X-ray crystallography in the full-length protein. Here, we determined an NMR structure of the isolated C-terminal domain of the Aspergillus nidulans mt TyrRS, which is closely related to but smaller than CYT-18's. The structure shows an S4 fold like that of bacterial TyrRSs, but with novel features, including three Pezizomycontia-specific insertions. (15)N-(1)H two-dimensional NMR showed that C-terminal domains of the full-length A. nidulans and Geobacillus stearothermophilus synthetases do not tumble independently in solution, suggesting restricted orientations. Modeling onto a CYT-18/group I intron cocrystal structure indicates that the C-terminal domains of both subunits of the homodimeric protein bind different ends of the intron RNA, with one C-terminal domain having to undergo a large shift on its flexible linker to bind tRNA(Tyr) or the intron RNA on either side of the catalytic domain. The modeling suggests that the C-terminal domain acts together with the N-terminal domain to clamp parts of the intron's catalytic core, that at least one C-terminal domain insertion functions in group I intron binding, and that some C-terminal domain regions bind both tRNA(Tyr) and group I intron RNAs.