BackgroundThe 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.ResultsHere 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.ConclusionsThe 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.

DNA has proved to be a versatile material for the rational design and assembly of nanometer scale objects. Here we report the crystal structure of a continuous three-dimensional DNA lattice formed by the self-assembly of a DNA 13-mer. The structure consists of stacked layers of parallel helices with adjacent layers linked through parallel-stranded base pairing. The hexagonal lattice geometry contains solvent channels that appear large enough to allow 3'-linked guest molecules into the crystal. We have successfully used these parallel base pairs to design and produce crystals with greatly enlarged solvent channels. This lattice may have applications as a molecular scaffold for structure determination of guest molecules, as a molecular sieve, or in the assembly of molecular electronics. Predictable non-Watson-Crick base pairs, like those described here, may present a new tool in structural DNA nanotechnology.

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.

The bifunctional Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) both aminoacylates mitochondrial tRNA Tyr and acts as a structure-stabilizing splicing cofactor for group I introns. Previous studies showed that CYT-18 has distinct tRNA Tyr and group I intron-binding sites, with the latter formed by three small ''insertions'' in the nucleotide-binding fold and other structural adaptations compared with nonsplicing bacterial tyrosyl-tRNA synthetases. Here, analysis of genomic sequences shows that mitochondrial tyrosyl-tRNA synthetases with structural adaptations similar to CYT-18's are uniquely characteristic of fungi belonging to the subphylum Pezizomycotina, and biochemical assays confirm group I intron splicing activity for the enzymes from several of these organisms, including Aspergillus nidulans and the human pathogens Coccidioides posadasii and Histoplasma capsulatum. By combining multiple sequence alignments with a previously determined cocrystal structure of a CYT-18/group I intron RNA complex, we identify conserved features of the Pezizomycotina enzymes related to group I intron and tRNA interactions. Our results suggest that mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity evolved during or after the divergence of the fungal subphyla Pezizomycotina and Saccharomycotina by a mechanism involving the concerted differentiation of preexisting protein loop regions. The unique group I intron splicing activity of these fungal enzymes may provide a new target for antifungal drugs.aminoacyl-tRNA synthetase ͉ medical mycology ͉ ribozyme ͉ RNA splicing ͉ protein structure function T he Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) is one of a number of proteins that adapted to function in splicing autocatalytic group I and group II introns in different organisms (1-3). One class of these proteins, splicing factors, functions by binding specifically to the intron RNAs and stabilizing their catalytically active RNA structure for efficient splicing in vivo. Groups I and II intron splicing factors differ among organisms and are commonly host proteins that have or had some other functions. Most promote the splicing of only one or a small number of structurally related introns, but CYT-18 is an exception, because it is able to splice diverse group I introns from N. crassa and other organisms (4-6). The idiosyncratic nature of group I and II intron splicing factors suggests that they adapted to function in splicing relatively recently in evolution, after the dispersal of the introns as mobile elements (2, 3).The CYT-18 protein was identified genetically in a screen for splicing-deficient mutants and shown to function in splicing three group I introns in N. crassa mitochondria: the mitochondrial (mt) large subunit rRNA (mtLSU) intron, ND1-I1, and cob-I2 (7,8). Biochemical studies showed that purified recombinant CYT-18 by itself stimulates group I intron splicing in vitro, that it recognizes conserved secondary and tertiary structure f...

We determined a 1.95 A X-ray crystal structure of a C-terminally truncated Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) that functions in splicing group I introns. CYT-18's nucleotide binding fold and intermediate alpha-helical domains superimpose on those of bacterial TyrRSs, except for an N-terminal extension and two small insertions not found in nonsplicing bacterial enzymes. These additions surround the cyt-18-1 mutation site and are sites of suppressor mutations that restore splicing, but not synthetase activity. Highly constrained models based on directed hydroxyl radical cleavage assays show that the group I intron binds at a site formed in part by the three additions on the nucleotide binding fold surface opposite that which binds tRNATyr. Our results show how essential proteins can progressively evolve new functions.

In the Escherichia coli periplasm, the formation of protein disulfide bonds is catalyzed by DsbA and DsbC. DsbA is a monomer that is maintained in a fully oxidized state by the membrane enzyme DsbB, whereas DsbC is a dimer that is kept reduced by a second membrane protein, DsbD. Although the catalytic regions of DsbA and DsbC are composed of structurally homologous thioredoxin motif domains, DsbA serves only as an oxidase in vivo, whereas DsbC catalyzes disulfide reduction and isomerization and also exhibits significant chaperone activity. To reconcile the distinct catalytic activities of DsbC and DsbA, we constructed a series of chimeras comprising of the dimerization domain of DsbC, with or without the adjacent ␣-helical linker region, fused either to the first, second, third, or fifth residue of intact DsbA or to thioredoxin. The chimeras fully substituted for DsbC in disulfide-bond rearrangement and also were able to restore protein oxidation in a dsbA background. Remarkably, the chimeras could serve as a single catalyst for both disulfide-bond formation and rearrangement, thus reconciling the kinetically competing DsbBDsbA and DsbD-DsbC pathways. This property appeared to depend on the orientation of the DsbA active-site cysteines with respect to the DsbC dimerization domain. In vitro, the chimeras had high chaperone activity and significant reductase activity but only 15-22% of the disulfide-isomerization activity of DsbC, suggesting that rearrangement of nonnative disulfides may be mediated primarily by cycles of random reduction and reoxidation. D isulfide bonds play a key role in the folding process of many secretory and membrane proteins. A series of cysteine thiol:disulfide oxidoreductases have evolved in both eukaryotic and prokaryotic systems to catalyze this critical cellular process. In particular, the Dsb family of the Escherichia coli periplasm consists of two distinct pathways: DsbA-DsbB and DsbC͞DsbG-DsbD. These pathways are involved in the formation of disulfides and the rearrangement of incorrectly formed bonds, respectively (1, 2). The extreme oxidizing nature of DsbA mediates rapid oxidation of substrate cysteines and, therefore, results in the formation of nonnative disulfides, which are in turn rearranged by DsbC and, to a lesser extent, DsbG. Despite the strong oxidizing environment of the periplasmic space, DsbC and DsbG have to be maintained in a reduced state to be able to catalyze the rearrangement of nonnative disulfide bonds (1, 2).DsbA and DsbC are found in entirely oxidized and reduced states, respectively (3). DsbA is recycled by the membrane protein DsbB, which then passes its electrons to the quinones and the respiratory chain, whereas DsbC is maintained in the reduced state by another membrane protein, DsbD, by means of an electrontransfer relay that involves thioredoxin (TrxA) and TrxA reductase in the cytoplasm (4). Remarkably, the Dsb machinery has evolved to capitalize on a strong thiol oxidant (DsbA) and a strong thiol reductant (DsbC) that do not appear to exchange ele...

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.

DNA has proved to be a successful nanoscale building block because of its inherent programmability and its predictable structural features. One long-standing goal of DNA nanotechnology has been the rational design and assembly of three-dimensional (3D) DNA crystals for use as molecular scaffolds, in molecular electronics assembly, and as molecular sieves. Here we demonstrate that rationally designed 3D DNA crystals with mesoporous features can function as molecular sieves by selectively adsorbing proteins based on size.