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Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199774


Over a thousand nucleus-encoded mitochondrial proteins are imported from the cytoplasm; however, mitochondrial (mt) DNA encodes for a small number of critical proteins and the entire suite of mt:tRNAs responsible for translating these proteins. Mitochondrial RNase P (mtRNase P) is a three-protein complex responsible for cleaving and processing the 5'-end of mt:tRNAs. Mutations in any of the three proteins can cause mitochondrial disease, as well as mutations in mitochondrial DNA. Great strides have been made in understanding the enzymology of mtRNase P; however, how the loss of each protein causes mitochondrial dysfunction and abnormal mt:tRNA processing in vivo has not been examined in detail. Here, we used Drosophila genetics to selectively remove each member of the complex in order to assess their specific contributions to mt:tRNA cleavage. Using this powerful model, we find differential effects on cleavage depending on which complex member is lost and which mt:tRNA is being processed. These data revealed in vivo subtleties of mtRNase P function that could improve understanding of human diseases.

Mitocôndrias/enzimologia , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Ribonuclease P/metabolismo , Alelos , Animais , Drosophila melanogaster/genética , Mitocôndrias/patologia , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo
J Biol Chem ; 291(22): 11911-27, 2016 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-27036943


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.

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/genética
Acta Crystallogr D Biol Crystallogr ; 71(Pt 12): 2471-8, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26627654


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.

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 , Termodinâmica
Biopolymers ; 103(11): 618-26, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26015367


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.

DNA/química , Cristalografia por Raios X , Conformação de Ácido Nucleico , Oligonucleotídeos/química