RESUMO
High fidelity tRNA aminoacylation by aminoacyl-tRNA synthetases is essential for cell viability. ProXp-ala is a trans-editing protein that is present in all three domains of life and is responsible for hydrolyzing mischarged Ala-tRNAPro and preventing mistranslation of proline codons. Previous studies have shown that, like bacterial prolyl-tRNA synthetase, Caulobacter crescentus ProXp-ala recognizes the unique C1:G72 terminal base pair of the tRNAPro acceptor stem, helping to ensure deacylation of Ala-tRNAPro but not Ala-tRNAAla. The structural basis for C1:G72 recognition by ProXp-ala is still unknown and was investigated here. NMR spectroscopy, binding, and activity assays revealed two conserved residues, K50 and R80, that likely interact with the first base pair, stabilizing the initial protein-RNA encounter complex. Modeling studies are consistent with direct interaction between R80 and the major groove of G72. A third key contact between A76 of tRNAPro and K45 of ProXp-ala was essential for binding and accommodating the CCA-3' end in the active site. We also demonstrated the essential role that the 2'OH of A76 plays in catalysis. Eukaryotic ProXp-ala proteins recognize the same acceptor stem positions as their bacterial counterparts, albeit with different nucleotide base identities. ProXp-ala is encoded in some human pathogens; thus, these results have the potential to inform new antibiotic drug design.
Assuntos
Aminoacil-tRNA Sintetases , RNA de Transferência de Prolina , Humanos , RNA de Transferência de Prolina/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Prolina/química , Aminoacilação de RNA de Transferência , Códon , Domínio CatalíticoRESUMO
Aminoacyl-tRNA synthetases (ARSs) catalyze the charging of specific amino acids onto cognate tRNAs, an essential process for protein synthesis. Mutations in ARSs are frequently associated with a variety of human diseases. The human EPRS1 gene encodes a bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) with two catalytic cores and appended domains that contribute to nontranslational functions. In this study, we report compound heterozygous mutations in EPRS1, which lead to amino acid substitutions P14R and E205G in two patients with diabetes and bone diseases. While neither mutation affects tRNA binding or association of EPRS with the multisynthetase complex, E205G in the glutamyl-tRNA synthetase (ERS) region of EPRS is defective in amino acid activation and tRNAGlu charging. The P14R mutation induces a conformational change and altered tRNA charging kinetics in vitro. We propose that the altered catalytic activity and conformational changes in the EPRS variants sensitize patient cells to stress, triggering an increased integrated stress response (ISR) that diminishes cell viability. Indeed, patient-derived cells expressing the compound heterozygous EPRS show heightened induction of the ISR, suggestive of disruptions in protein homeostasis. These results have important implications for understanding ARS-associated human disease mechanisms and development of new therapeutics.
Assuntos
Doenças Ósseas , Diabetes Mellitus , Doenças Genéticas Inatas , Glutamato-tRNA Ligase , Mutação de Sentido Incorreto , Estresse Fisiológico/genética , Substituição de Aminoácidos , Doenças Ósseas/enzimologia , Doenças Ósseas/genética , Diabetes Mellitus/enzimologia , Diabetes Mellitus/genética , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/genética , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismo , Células HEK293 , Humanos , MasculinoRESUMO
The recent identification and development of RNA-guided enzymes for programmable cleavage of target nucleic acids offers exciting possibilities for both therapeutic and biotechnological applications. However, critical challenges such as expensive guide RNAs and inability to predict the efficiency of target recognition, especially for highly-structured RNAs, remain to be addressed. Here, we introduce a programmable RNA restriction enzyme, based on a budding yeast Argonaute (AGO), programmed with cost-effective 23-nucleotide (nt) single-stranded DNAs as guides. DNA guides offer the advantage that diverse sequences can be easily designed and purchased, enabling high-throughput screening to identify optimal recognition sites in the target RNA. Using this DNA-induced slicing complex (DISC) programmed with 11 different guide DNAs designed to span the sequence, sites of cleavage were identified in the 352-nt human immunodeficiency virus type 1 5'-untranslated region. This assay, coupled with primer extension and capillary electrophoresis, allows detection and relative quantification of all DISC-cleavage sites simultaneously in a single reaction. Comparison between DISC cleavage and RNase H cleavage reveals that DISC not only cleaves solvent-exposed sites, but also sites that become more accessible upon DISC binding. This study demonstrates the advantages of the DISC system for programmable cleavage of highly-structured, functional RNAs.
Assuntos
Proteínas Argonautas/metabolismo , DNA de Cadeia Simples/metabolismo , RNA/metabolismo , Ribonucleases/metabolismo , Regiões 5' não Traduzidas/genética , Sequência de Bases , Sítios de Ligação/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , HIV-1/genética , Humanos , Conformação de Ácido Nucleico , RNA/química , RNA/genética , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Especificidade por SubstratoRESUMO
Molecular sieves ensure proper pairing of tRNAs and amino acids during aminoacyl-tRNA biosynthesis, thereby avoiding detrimental effects of mistranslation on cell growth and viability. Mischarging errors are often corrected through the activity of specialized editing domains present in some aminoacyl-tRNA synthetases or via single-domain trans-editing proteins. ProXp-ala is a ubiquitous trans-editing enzyme that edits Ala-tRNAPro, the product of Ala mischarging by prolyl-tRNA synthetase, although the structural basis for discrimination between correctly charged Pro-tRNAPro and mischarged Ala-tRNAAla is unclear. Deacylation assays using substrate analogs reveal that size discrimination is only one component of selectivity. We used NMR spectroscopy and sequence conservation to guide extensive site-directed mutagenesis of Caulobacter crescentus ProXp-ala, along with binding and deacylation assays to map specificity determinants. Chemical shift perturbations induced by an uncharged tRNAPro acceptor stem mimic, microhelixPro, or a nonhydrolyzable mischarged Ala-microhelixPro substrate analog identified residues important for binding and deacylation. Backbone 15N NMR relaxation experiments revealed dynamics for a helix flanking the substrate binding site in free ProXp-ala, likely reflecting sampling of open and closed conformations. Dynamics persist on binding to the uncharged microhelix, but are attenuated when the stably mischarged analog is bound. Computational docking and molecular dynamics simulations provide structural context for these findings and predict a role for the substrate primary α-amine group in substrate recognition. Overall, our results illuminate strategies used by a trans-editing domain to ensure acceptance of only mischarged Ala-tRNAPro, including conformational selection by a dynamic helix, size-based exclusion, and optimal positioning of substrate chemical groups.
Assuntos
Aminoacil-tRNA Sintetases/genética , Proteínas de Bactérias/genética , Caulobacter crescentus/genética , Biossíntese de Proteínas/genética , RNA de Transferência de Prolina/genética , Aminoácidos/genética , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Caulobacter crescentus/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Conformação Proteica , Edição de RNA , RNA de Transferência de Prolina/química , RNA de Transferência de Prolina/metabolismo , Especificidade por SubstratoRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is the first retrovirus that has conclusively been shown to cause human diseases. In HIV-1, specific interactions between the nucleocapsid (NC) domain of the Gag protein and genomic RNA (gRNA) mediate gRNA dimerization and selective packaging; however, the mechanism for gRNA packaging in HTLV-1, a deltaretrovirus, is unclear. In other deltaretroviruses, the matrix (MA) and NC domains of Gag are both involved in gRNA packaging, but MA binds nucleic acids with higher affinity and has more robust chaperone activity, suggesting that this domain may play a primary role. Here, we show that the MA domain of HTLV-1, but not the NC domain, binds short hairpin RNAs derived from the putative gRNA packaging signal. RNA probing of the HTLV-1 5' leader and cross-linking studies revealed that the primer-binding site and a region within the putative packaging signal form stable hairpins that interact with MA. In addition to a previously identified palindromic dimerization initiation site (DIS), we identified a new DIS in HTLV-1 gRNA and found that both palindromic sequences bind specifically the NC domain. Surprisingly, a mutant partially defective in dimer formation in vitro exhibited a significant increase in RNA packaging into HTLV-1-like particles, suggesting that efficient RNA dimerization may not be strictly required for RNA packaging in HTLV-1. Moreover, the lifecycle of HTLV-1 and other deltaretroviruses may be characterized by NC and MA functions that are distinct from those of the corresponding HIV-1 proteins, but together provide the functions required for viral replication.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano/química , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Dimerização , Proteínas do Vírus da Imunodeficiência Humana/química , Proteínas do Vírus da Imunodeficiência Humana/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Nucleocapsídeo/genética , Proteínas de Ligação a RNA/fisiologia , Replicação ViralRESUMO
Chemical and enzymatic probing of RNA secondary structure and RNA/protein interactions provides the basis for understanding the functions of structured RNAs. However, the ability to rapidly perform such experiments using capillary electrophoresis has been hampered by relatively labor-intensive data analysis software. While these computationally robust programs have been shown to calculate residue-specific reactivities to a high degree of accuracy, they often require time-consuming manual intervention and lack the ability to be easily modified by users. To alleviate these issues, RiboCAT (Ribonucleic acid capillary-electrophoresis analysis tool) was developed as a user-friendly, Microsoft Excel-based tool that reduces the need for manual intervention, thereby significantly shortening the time required for data analysis. Features of this tool include (i) the use of an Excel platform, (ii) a method of intercapillary signal alignment using internal size standards, (iii) a peak-sharpening algorithm to more accurately identify peaks, and (iv) an open architecture allowing for simple user intervention. Furthermore, a complementary tool, RiboDOG (RiboCAT data output generator) was designed to facilitate the comparison of multiple data sets, highlighting potential inconsistencies and inaccuracies that may have occurred during analysis. Using these new tools, the secondary structure of the HIV-1 5' untranslated region (5'UTR) was determined using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), matching the results of previous work.
Assuntos
Algoritmos , Eletroforese Capilar/estatística & dados numéricos , Sondas de Ácido Nucleico/análise , RNA Viral/análise , Software , Regiões 5' não Traduzidas , Acilação , Pareamento de Bases , Sequência de Bases , HIV-1/química , HIV-1/genética , Conformação de Ácido Nucleico , Sondas de Ácido Nucleico/química , RNA Viral/químicaRESUMO
Accuracy in protein biosynthesis is maintained through multiple pathways, with a critical checkpoint occurring at the tRNA aminoacylation step catalyzed by aminoacyl-tRNA synthetases (ARSs). In addition to the editing functions inherent to some synthetases, single-domain trans-editing factors, which are structurally homologous to ARS editing domains, have evolved as alternative mechanisms to correct mistakes in aminoacyl-tRNA synthesis. To date, ARS-like trans-editing domains have been shown to act on specific tRNAs that are mischarged with genetically encoded amino acids. However, structurally related non-protein amino acids are ubiquitous in cells and threaten the proteome. Here, we show that a previously uncharacterized homolog of the bacterial prolyl-tRNA synthetase (ProRS) editing domain edits a known ProRS aminoacylation error, Ala-tRNAPro, but displays even more robust editing of tRNAs misaminoacylated with the non-protein amino acid α-aminobutyrate (2-aminobutyrate, Abu) in vitro and in vivo. Our results indicate that editing by trans-editing domains such as ProXp-x studied here may offer advantages to cells, especially under environmental conditions where concentrations of non-protein amino acids may challenge the substrate specificity of ARSs.
Assuntos
Aminoacil-tRNA Sintetases/genética , Aminobutiratos/metabolismo , Prolina/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência de Prolina/genética , Aminoacilação de RNA de Transferência , Alanina/genética , Alanina/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Aminobutiratos/química , Anticódon/química , Anticódon/metabolismo , Sítios de Ligação , Códon/química , Códon/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Simulação de Acoplamento Molecular , Mutação , Conformação de Ácido Nucleico , Prolina/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , RNA de Transferência de Prolina/química , RNA de Transferência de Prolina/metabolismo , Rodopseudomonas/genética , Rodopseudomonas/metabolismo , Especificidade por SubstratoRESUMO
In addition to their role in correctly attaching specific amino acids to cognate tRNAs, aminoacyl-tRNA synthetases (aaRS) have been found to possess many alternative functions and often bind to and act on other nucleic acids. In contrast to the well-defined 3D structure of tRNA, the structures of many of the other RNAs recognized by aaRSs have not been solved. Despite advances in the use of X-ray crystallography (XRC), nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) for structural characterization of biomolecules, significant challenges to solving RNA structures still exist. Recently, small-angle X-ray scattering (SAXS) has been increasingly employed to characterize the 3D structures of RNAs and RNA-protein complexes. SAXS is capable of providing low-resolution tertiary structure information under physiological conditions and with less intensive sample preparation and data analysis requirements than XRC, NMR and cryo-EM. In this article, we describe best practices involved in the process of RNA and RNA-protein sample preparation, SAXS data collection, data analysis, and structural model building.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Escherichia coli/genética , Dobramento de RNA , RNA de Transferência Aminoácido-Específico/química , Aminoacilação de RNA de Transferência , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/genética , Cromatografia em Gel , Escherichia coli/metabolismo , Modelos Moleculares , Eletroforese em Gel de Poliacrilamida Nativa , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , RNA de Transferência Aminoácido-Específico/genética , RNA de Transferência Aminoácido-Específico/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The most conserved region of the HIV type 1 (HIV-1) genome, the â¼335-nt 5' UTR, is characterized by functional stem loop domains responsible for regulating the viral life cycle. Despite the indispensable nature of this region of the genome in HIV-1 replication, 3D structures of multihairpin domains of the 5' UTR remain unknown. Using small-angle X-ray scattering and molecular dynamics simulations, we generated structural models of the transactivation (TAR)/polyadenylation (polyA), primer-binding site (PBS), and Psi-packaging domains. TAR and polyA form extended, coaxially stacked hairpins, consistent with their high stability and contribution to the pausing of reverse transcription. The Psi domain is extended, with each stem loop exposed for interactions with binding partners. The PBS domain adopts a bent conformation resembling the shape of a tRNA in apo and primer-annealed states. These results provide a structural basis for understanding several key molecular mechanisms underlying HIV-1 replication.
Assuntos
Regiões 5' não Traduzidas/genética , HIV-1/química , Modelos Moleculares , Pareamento de Bases , Sequência de Bases , Cromatografia em Gel , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , RNA de Transferência/química , Espalhamento a Baixo ÂnguloRESUMO
Codon use among the three domains of life is not confined to the universal genetic code. With only 22 tRNA genes in mammalian mitochondria, exceptions from the universal code are necessary for proper translation. A particularly interesting deviation is the decoding of the isoleucine AUA codon as methionine by the one mitochondrial-encoded tRNA(Met). This tRNA decodes AUA and AUG in both the A- and P-sites of the metazoan mitochondrial ribosome. Enrichment of posttranscriptional modifications is a commonly appropriated mechanism for modulating decoding rules, enabling some tRNA functions while restraining others. In this case, a modification of cytidine, 5-formylcytidine (f(5)C), at the wobble position-34 of human mitochondrial tRNA(f5CAU)(Met) (hmtRNA(f5CAU)(Met)) enables expanded decoding of AUA, resulting in a deviation in the genetic code. Visualization of the codonâ¢anticodon interaction by X-ray crystallography revealed that recognition of both A and G at the third position of the codon occurs in the canonical Watson-Crick geometry. A modification-dependent shift in the tautomeric equilibrium toward the rare imino-oxo tautomer of cytidine stabilizes the f(5)C34â¢A base pair geometry with two hydrogen bonds.
Assuntos
Códon/química , Códon/genética , RNA de Transferência/química , RNA de Transferência/genética , Pareamento de Bases , Cristalografia por Raios X , Citidina/análogos & derivados , Citidina/química , Humanos , Isomerismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/genéticaRESUMO
Since its inception in 1994, The RNA Modification Database (RNAMDB, http://rna-mdb.cas.albany.edu/RNAmods/) has served as a focal point for information pertaining to naturally occurring RNA modifications. In its current state, the database employs an easy-to-use, searchable interface for obtaining detailed data on the 109 currently known RNA modifications. Each entry provides the chemical structure, common name and symbol, elemental composition and mass, CA registry numbers and index name, phylogenetic source, type of RNA species in which it is found, and references to the first reported structure determination and synthesis. Though newly transferred in its entirety to The RNA Institute, the RNAMDB continues to grow with two notable additions, agmatidine and 8-methyladenosine, appended in the last year. The RNA Modification Database is staying up-to-date with significant improvements being prepared for inclusion within the next year and the following year. The expanded future role of The RNA Modification Database will be to serve as a primary information portal for researchers across the entire spectrum of RNA-related research.
Assuntos
Bases de Dados de Ácidos Nucleicos , Processamento Pós-Transcricional do RNA , RNA/química , Adenosina/análogos & derivados , Adenosina/química , Citidina/análogos & derivados , Citidina/químicaRESUMO
The antiviral component of Paxlovid, nirmatrelvir (NIR), forms a covalent bond with Cys145 of SARS-CoV-2 nsp5. To explore NIR resistance we designed mutations to impair binding of NIR over substrate. Using 12 Omicron (BA.1) and WA.1 SARS-CoV-2 replicons, cell-based complementation and enzymatic assays, we showed that in both strains, E166V imparted high NIR resistance (â¼55-fold), with major decrease in WA1 replicon fitness (â¼20-fold), but not BA.1 (â¼2-fold). WA1 replicon fitness was restored by L50F. These differences may contribute to a potentially lower barrier to resistance in Omicron than WA1. E166V is rare in untreated patients, albeit more prevalent in paxlovid-treated EPIC-HR clinical trial patients. Importantly, NIR-resistant replicons with E166V or E166V/L50F remained susceptible to a) the flexible GC376, and b) PF-00835231, which forms additional interactions. Molecular dynamics simulations show steric clashes between the rigid and bulky NIR t-butyl and ß-branched V166 distancing the NIR warhead from its Cys145 target. In contrast, GC376, through "wiggling and jiggling" accommodates V166 and still covalently binds Cys145. PF-00835231 uses its strategically positioned methoxy-indole to form a ß-sheet and overcome E166V. Drug design based on strategic flexibility and main chain-targeting may help develop second-generation nsp5-targeting antivirals efficient against NIR-resistant viruses.
RESUMO
Interactions between lysyl-tRNA synthetase (LysRS) and HIV-1 Gag facilitate selective packaging of the HIV-1 reverse transcription primer, tRNALys3. During HIV-1 infection, LysRS is phosphorylated at S207, released from a multi-aminoacyl-tRNA synthetase complex and packaged into progeny virions. LysRS is critical for proper targeting of tRNALys3 to the primer-binding site (PBS) by specifically binding a PBS-adjacent tRNA-like element (TLE), which promotes release of the tRNA proximal to the PBS. However, whether LysRS phosphorylation plays a role in this process remains unknown. Here, we used a combination of binding assays, RNA chemical probing, and small-angle X-ray scattering to show that both wild-type (WT) and a phosphomimetic S207D LysRS mutant bind similarly to the HIV-1 genomic RNA (gRNA) 5'UTR via direct interactions with the TLE and stem loop 1 (SL1) and have a modest preference for binding dimeric gRNA. Unlike WT, S207D LysRS bound in an open conformation and increased the dynamics of both the PBS region and SL1. A new working model is proposed wherein a dimeric phosphorylated LysRS/tRNA complex binds to a gRNA dimer to facilitate tRNA primer release and placement onto the PBS. Future anti-viral strategies that prevent this host factor-gRNA interaction are envisioned.
Assuntos
Soropositividade para HIV , HIV-1 , Lisina-tRNA Ligase , Regiões 5' não Traduzidas , Soropositividade para HIV/genética , HIV-1/genética , HIV-1/metabolismo , Humanos , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/genética , Conformação de Ácido Nucleico , RNA Guia de Cinetoplastídeos , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Human mitochondrial methionine transfer RNA (hmtRNA(Met)(CAU)) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position-34 (f(5)C(34)). The role of this modification in (hmtRNA(Met)(CAU)) for the decoding of AUA, as well as AUG, in both the peptidyl- and aminoacyl-sites of the ribosome in either chain initiation or chain elongation is still unknown. We report the first synthesis and analyses of the tRNA's anticodon stem and loop domain containing the 5-formylcytidine modification. The modification contributes to the tRNA's anticodon domain structure, thermodynamic properties and its ability to bind codons AUA and AUG in translational initiation and elongation.
Assuntos
Anticódon/química , Citidina/análogos & derivados , Biossíntese de Proteínas , RNA de Transferência de Metionina/química , RNA/química , Sequência de Bases , Códon/metabolismo , Citidina/química , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mitocondrial , RNA de Transferência de Metionina/síntese química , RNA de Transferência de Metionina/metabolismo , TermodinâmicaRESUMO
Three of six arginine codons (CGU, CGC, and CGA) are decoded by two Escherichia coli tRNAArg isoacceptors. The anticodon stem and loop (ASL) domains of tRNAArg1 and tRNAArg2 both contain inosine and 2-methyladenosine modifications at positions 34 (I34) and 37 (m2A37). tRNAArg1 is also modified from cytidine to 2-thiocytidine at position 32 (s2C32). The s2C32 modification is known to negate wobble codon recognition of the rare CGA codon by an unknown mechanism, while still allowing decoding of CGU and CGC. Substitution of s2C32 for C32 in the Saccharomyces cerevisiae tRNAIleIAU anticodon stem and loop domain (ASL) negates wobble decoding of its synonymous A-ending codon, suggesting that this function of s2C at position 32 is a generalizable property. X-ray crystal structures of variously modified ASLArg1ICG and ASLArg2ICG constructs bound to cognate and wobble codons on the ribosome revealed the disruption of a C32-A38 cross-loop interaction but failed to fully explain the means by which s2C32 restricts I34 wobbling. Computational studies revealed that the adoption of a spatially broad inosine-adenosine base pair at the wobble position of the codon cannot be maintained simultaneously with the canonical ASL U-turn motif. C32-A38 cross-loop interactions are required for stability of the anticodon/codon interaction in the ribosomal A-site.
Assuntos
Códon/genética , Citidina/análogos & derivados , RNA de Transferência/metabolismo , Biologia Computacional , Cristalografia por Raios X , Citidina/metabolismo , Inosina/metabolismo , Nucleosídeos/metabolismo , RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , TermodinâmicaRESUMO
Bovine leukemia virus (BLV) is a deltaretrovirus that infects domestic cattle. The structural protein Gag, found in all retroviruses, is a polyprotein comprising three major functional domains: matrix (MA), capsid (CA), and nucleocapsid (NC). Previous studies have shown that both mature BLV MA and NC are able to bind to nucleic acids; however, the viral assembly process and packaging of viral genomic RNA requires full-length Gag to produce infectious particles. Compared to lentiviruses, little is known about the structure of the Gag polyprotein of deltaretroviruses. In this work, structural models of full-length BLV Gag and Gag lacking the MA domain were generated based on previous structural data of individual domains, homology modeling, and flexible fitting to SAXS data using molecular dynamics. The models were used in molecular dynamic simulations to determine the relative mobility of the protein backbone. Functional annealing assays revealed the role of MA in the nucleic acid chaperone activity of BLV Gag. Our results show that full-length BLV Gag has an elongated rod-shaped structure that is relatively rigid, with the exception of the linker between the MA and CA domains. Deletion of the MA domain maintains the elongated structure but alters the rate of BLV Gag-facilitated annealing of two complementary nucleic acids. These data are consistent with a role for the MA domain of retroviral Gag proteins in modulating nucleic acid binding and chaperone activity. IMPORTANCE: BLV is a retrovirus that is found worldwide in domestic cattle. Since BLV infection has serious implications for agriculture, and given its similarities to human retroviruses such as HTLV-1, the development of an effective treatment would have numerous benefits. The Gag polyprotein exists in all retroviruses and is a key player in viral assembly. However, the full-length structure of Gag from any virus has yet to be elucidated at high resolution. This study provides structural data for BLV Gag and could be a starting point for modeling Gag-small molecule interactions with the ultimate goal of developing of a new class of pharmaceuticals.
Assuntos
Bovinos/virologia , Leucose Enzoótica Bovina/virologia , Produtos do Gene gag/química , Vírus da Leucemia Bovina/química , Animais , Modelos Moleculares , Conformação Proteica , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The emergence of multidrug-resistant bacteria necessitates the identification of unique targets of intervention and compounds that inhibit their function. Gram-positive bacteria use a well-conserved tRNA-responsive transcriptional regulatory element in mRNAs, known as the T-box, to regulate the transcription of multiple operons that control amino acid metabolism. T-box regulatory elements are found only in the 5'-untranslated region (UTR) of mRNAs of Gram-positive bacteria, not Gram-negative bacteria or the human host. Using the structure of the 5'UTR sequence of the Bacillus subtilis tyrosyl-tRNA synthetase mRNA T-box as a model, inâ silico docking of 305 000 small compounds initially yielded 700 as potential binders that could inhibit the binding of the tRNA ligand. A single family of compounds inhibited the growth of Gram-positive bacteria, but not Gram-negative bacteria, including drug-resistant clinical isolates at minimum inhibitory concentrations (MIC 16-64â µg mL-1 ). Resistance developed at an extremely low mutational frequency (1.21×10-10 ). At 4â µg mL-1 , the parent compound PKZ18 significantly inhibited inâ vivo transcription of glycyl-tRNA synthetase mRNA. PKZ18 also inhibited inâ vivo translation of the S.â aureus threonyl-tRNA synthetase protein. PKZ18 bound to the Specifier Loop inâ vitro (Kd ≈24â µm). Its core chemistry necessary for antibacterial activity has been identified. These findings support the T-box regulatory mechanism as a new target for antibiotic discovery that may impede the emergence of resistance.
Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , RNA de Transferência/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Antibacterianos/química , Bactérias Gram-Positivas/genética , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , RNA Mensageiro/genética , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Two copies of unspliced human immunodeficiency virus (HIV)-1 genomic RNA (gRNA) are preferentially selected for packaging by the group-specific antigen (Gag) polyprotein into progeny virions as a dimer during the late stages of the viral lifecycle. Elucidating the RNA features responsible for selective recognition of the full-length gRNA in the presence of an abundance of other cellular RNAs and spliced viral RNAs remains an area of intense research. The recent nuclear magnetic resonance (NMR) structure by Keane et al. [1] expands upon previous efforts to determine the conformation of the HIV-1 RNA packaging signal. The data support a secondary structure wherein sequences that constitute the major splice donor site are sequestered through base pairing, and a tertiary structure that adopts a tandem 3-way junction motif that exposes the dimerization initiation site and unpaired guanosines for specific recognition by Gag. While it remains to be established whether this structure is conserved in the context of larger RNA constructs or in the dimer, this study serves as the basis for characterizing large RNA structures using novel NMR techniques, and as a major advance toward understanding how the HIV-1 gRNA is selectively packaged.
Assuntos
HIV-1/fisiologia , RNA Viral/química , RNA Viral/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Pareamento de Bases , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
The field of viral molecular biology has reached a precipice for which pioneering studies on the structure of viral RNAs are beginning to bridge the gap. It has become clear that viral genomic RNAs are not simply carriers of hereditary information, but rather are active players in many critical stages during replication. Indeed, functions such as cap-independent translation initiation mechanisms are, in some cases, primarily driven by RNA structural determinants. Other stages including reverse transcription initiation in retroviruses, nuclear export and viral packaging are specifically dependent on the proper 3-dimensional folding of multiple RNA domains to recruit necessary viral and host factors required for activity. Furthermore, a large-scale conformational change within the 5'-untranslated region of HIV-1 has been proposed to regulate the temporal switch between viral protein synthesis and packaging. These RNA-dependent functions are necessary for replication of many human disease-causing viruses such as severe acute respiratory syndrome (SARS)-associated coronavirus, West Nile virus, and HIV-1. The potential for antiviral development is currently hindered by a poor understanding of RNA-driven molecular mechanisms, resulting from a lack of structural information on large RNAs and ribonucleoprotein complexes. Herein, we describe the recent progress that has been made on characterizing these large RNAs and provide brief descriptions of the techniques that will be at the forefront of future advances. Ongoing and future work will contribute to a more complete understanding of the lifecycles of retroviruses and RNA viruses and potentially lead to novel antiviral strategies.