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1.
Nucleic Acids Res ; 52(9): 5285-5300, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38366771

RESUMO

The signal recognition particle (SRP) is a critical component in protein sorting pathways in all domains of life. Human SRP contains six proteins bound to the 7S RNA and their structures and functions have been mostly elucidated. The SRP68/72 dimer is the largest SRP component and is essential for SRP function. Although the structures of the SRP68/72 RNA binding and dimerization domains have been previously reported, the structure and function of large portions of the SRP68/72 dimer remain unknown. Here, we analyse full-length SRP68/72 using cryo-EM and report that SRP68/72 depend on each other for stability and form an extended dimerization domain. This newly observed dimerization domain is both a protein- and RNA-binding domain. Comparative analysis with current structural models suggests that this dimerization domain undergoes dramatic translocation upon SRP docking onto SRP receptor and eventually comes close to the Alu domain. We propose that the SRP68/72 dimerization domain functions by binding and detaching the Alu domain and SRP9/14 from the ribosomal surface, thus releasing elongation arrest upon docking onto the ER membrane.


Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , Multimerização Proteica , Partícula de Reconhecimento de Sinal , Humanos , Sítios de Ligação , Ligação Proteica , Domínios Proteicos , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/metabolismo
2.
Proteins ; 92(3): 418-426, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37929701

RESUMO

Middle East respiratory syndrome coronavirus (MERS CoV) and severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) are RNA viruses from the Betacoronavirus family that cause serious respiratory illness in humans. One of the conserved non-structural proteins encoded for by the coronavirus family is non-structural protein 9 (nsp9). Nsp9 plays an important role in the RNA capping process of the viral genome, where it is covalently linked to viral RNA (known as RNAylation) by the conserved viral polymerase, nsp12. Nsp9 also directly binds to RNA; we have recently revealed a distinct RNA recognition interface in the SARS CoV-2 nsp9 by using a combination of nuclear magnetic resonance spectroscopy and biolayer interferometry. In this study, we have utilized a similar methodology to determine a structural model of RNA binding of the related MERS CoV. Based on these data, we uncover important similarities and differences to SARS CoV-2 nsp9 and other coronavirus nsp9 proteins. Our findings that replacing key RNA binding residues in MERS CoV nsp9 affects RNAylation efficiency indicate that recognition of RNA may play a role in the capping process of the virus.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , RNA/metabolismo
3.
Proteins ; 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38958516

RESUMO

The ongoing global pandemic of the coronavirus 2019 (COVID-19) disease is caused by the virus SARS-CoV-2, with very few highly effective antiviral treatments currently available. The machinery responsible for the replication and transcription of viral RNA during infection is made up of several important proteins. Two of these are nsp12, the catalytic subunit of the viral polymerase, and nsp9, a cofactor of nsp12 involved in the capping and priming of viral RNA. While several recent studies have determined the structural details of the interaction of nsp9 with nsp12 in the context of RNA capping, very few biochemical or biophysical details are currently available. In this study, we have used a combination of surface plasmon resonance (SPR) experiments, size exclusion chromatography (SEC) experiments, and biochemical assays to identify specific nsp9 residues that are critical for nsp12 binding as well as RNAylation, both of which are essential for the RNA capping process. Our data indicate that nsp9 dimerization is unlikely to play a significant functional role in the virus. We confirm that a set of recently discovered antiviral peptides inhibit nsp9-nsp12 interaction by specifically binding to nsp9; however, we find that these peptides do not impact RNAylation. In summary, our results have important implications for future drug discovery efforts to combat SARS-CoV-2 and any newly emerging coronaviruses.

4.
Nucleic Acids Res ; 50(5): 2889-2904, 2022 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-35150565

RESUMO

Regulated transcription termination provides an efficient and responsive means to control gene expression. In bacteria, rho-independent termination occurs through the formation of an intrinsic RNA terminator loop, which disrupts the RNA polymerase elongation complex, resulting in its dissociation from the DNA template. Bacteria have a number of pathways for overriding termination, one of which is the formation of mutually exclusive RNA motifs. ANTAR domains are a class of antiterminator that bind and stabilize dual hexaloop RNA motifs within the nascent RNA chain to prevent terminator loop formation. We have determined the structures of the dimeric ANTAR domain protein EutV, from Enterococcus faecialis, in the absence of and in complex with the dual hexaloop RNA target. The structures illustrate conformational changes that occur upon RNA binding and reveal that the molecular interactions between the ANTAR domains and RNA are restricted to a single hexaloop of the motif. An ANTAR domain dimer must contact each hexaloop of the dual hexaloop motif individually to prevent termination in eubacteria. Our findings thereby redefine the minimal ANTAR domain binding motif to a single hexaloop and revise the current model for ANTAR-mediated antitermination. These insights will inform and facilitate the discovery of novel ANTAR domain RNA targets.


Assuntos
Proteínas de Bactérias/química , Enterococcus faecalis/metabolismo , Proteínas de Ligação a RNA/química , Terminação da Transcrição Genética , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Bacteriano/genética , Regiões Terminadoras Genéticas , Transcrição Gênica
5.
J Biol Chem ; 297(4): 101165, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34487761

RESUMO

The bacterial insertion sequence (IS) IS26 mobilizes and disseminates antibiotic resistance genes. It differs from bacterial IS that have been studied to date as it exclusively forms cointegrates via either a copy-in (replicative) or a recently discovered targeted conservative mode. To investigate how the Tnp26 transposase recognizes the 14-bp terminal inverted repeats (TIRs) that bound the IS, amino acids in two domains in the N-terminal (amino acids M1-P56) region were replaced. These changes substantially reduced cointegration in both modes. Tnp26 was purified as a maltose-binding fusion protein and shown to bind specifically to dsDNA fragments that included an IS26 TIR. However, Tnp26 with an R49A or a W50A substitution in helix 3 of a predicted trihelical helix-turn-helix domain (amino acids I13-R53) or an F4A or F9A substitution replacing the conserved amino acids in a unique disordered N-terminal domain (amino acids M1-D12) did not bind. The N-terminal M1-P56 fragment also bound to the TIR but only at substantially higher concentrations, indicating that other parts of Tnp26 enhance the binding affinity. The binding site was confined to the internal part of the TIR, and a G to T nucleotide substitution in the TGT at positions 6 to 8 of the TIR that is conserved in most IS26 family members abolished binding of both Tnp26 (M1-M234) and Tnp26 M1-P56 fragment. These findings indicate that the helix-turn-helix and disordered domains of Tnp26 play a role in Tnp26-TIR complex formation. Both domains are conserved in all members of the IS26 family.


Assuntos
Elementos de DNA Transponíveis , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Sequências Repetidas Terminais , Transposases/química , Substituição de Aminoácidos , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação de Sentido Incorreto , Domínios Proteicos , Transposases/genética , Transposases/metabolismo
6.
Protein Expr Purif ; 198: 106121, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35640773

RESUMO

The Signal Recognition Particle (SRP) and the SRP receptor (SR) are responsible for protein targeting to the plasma membrane and the protein secretory pathway. Eukaryotic SRα, one of the two proteins that form the SR, is composed of the NG, MoRF and X domains. The SRα-NG domain is responsible for binding to SRP proteins such as SRP54, interacting with RNA, binding and hydrolysing GTP. The ability to produce folded SRα-NG is a prerequisite for structural studies directed towards a better understanding of its molecular mechanism and function, as well as in (counter-)screening assays for potential binders in the drug development pipeline. However, previously reported SRα-NG constructs and purification methods only used a truncated version, lacking the first N-terminal helix. This helix in other NG domains (e.g., SRP54) has been shown to be important for protein:protein interactions but its importance in SRα remains unknown. Here, we present the cloning as well as optimised expression and purification protocols of the whole SRα-NG domain including the first N-terminal helix. We have also expressed and purified isotopically labelled SRα-NG to facilitate Nuclear Magnetic Resonance (NMR) studies.


Assuntos
GTP Fosfo-Hidrolases , Partícula de Reconhecimento de Sinal , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Humanos , Ligação Proteica , Receptores Citoplasmáticos e Nucleares , Receptores de Peptídeos/química , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo
7.
Mol Cell ; 52(5): 643-54, 2013 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-24211265

RESUMO

The universally conserved signal recognition particle (SRP) system mediates the targeting of membrane proteins to the translocon in a multistep process controlled by GTP hydrolysis. Here we present the 2.6 Å crystal structure of the GTPase domains of the E. coli SRP protein (Ffh) and its receptor (FtsY) in complex with the tetraloop and the distal region of SRP-RNA, trapped in the activated state in presence of GDP:AlF4. The structure reveals the atomic details of FtsY recruitment and, together with biochemical experiments, pinpoints G83 as the key RNA residue that stimulates GTP hydrolysis. Insertion of G83 into the FtsY active site orients a single glutamate residue provided by Ffh (E277), triggering GTP hydrolysis and complex disassembly at the end of the targeting cycle. The complete conservation of the key residues of the SRP-RNA and the SRP protein implies that the suggested chemical mechanism of GTPase activation is applicable across all kingdoms.


Assuntos
Proteínas de Bactérias/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , RNA/genética , Receptores Citoplasmáticos e Nucleares/genética , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo , Compostos de Alumínio/farmacologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluoretos/farmacologia , Ativadores de GTP Fosfo-Hidrolase/farmacologia , Guanosina Difosfato/genética , Guanosina Difosfato/metabolismo , Hidrólise/efeitos dos fármacos , Dados de Sequência Molecular , RNA/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Análise de Sequência de DNA
8.
Proc Natl Acad Sci U S A ; 114(37): 9942-9947, 2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28847966

RESUMO

RNA has been found to interact with chromatin and modulate gene transcription. In human cells, little is known about how long noncoding RNAs (lncRNAs) interact with target loci in the context of chromatin. We find here, using the phosphatase and tensin homolog (PTEN) pseudogene as a model system, that antisense lncRNAs interact first with a 5' UTR-containing promoter-spanning transcript, which is then followed by the recruitment of DNA methyltransferase 3a (DNMT3a), ultimately resulting in the transcriptional and epigenetic control of gene expression. Moreover, we find that the lncRNA and promoter-spanning transcript interaction are based on a combination of structural and sequence components of the antisense lncRNA. These observations suggest, on the basis of this one example, that evolutionary pressures may be placed on RNA structure more so than sequence conservation. Collectively, the observations presented here suggest a much more complex and vibrant RNA regulatory world may be operative in the regulation of gene expression.


Assuntos
PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/fisiologia , Cromatina/genética , Montagem e Desmontagem da Cromatina , DNA Metiltransferase 3A , Éxons , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Pseudogenes , Elementos Reguladores de Transcrição/genética , Elementos Reguladores de Transcrição/fisiologia , Análise de Sequência de RNA/métodos , Homologia de Sequência
9.
J Struct Biol ; 208(3): 107387, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31520694

RESUMO

The bacterial signal recognition particle (SRP) receptor, FtsY, participates with the SRP in co-translation targeting of proteins. Multiple crystal structures of the NG domain of E. coli FtsYNG have been determined at high-resolution (1.22-1.88 Å), in the nucleotide-free (apo) form as well as bound to GDP and non-hydrolysable GTP analogues. The combination of high-resolution and multiple solved structures of FtsYNG in different states revealed a new sensor-relay system of this unique GTPase receptor. A nucleotide sensing function of the P-loop assists FtsYNG in nucleotide-binding and contributes to modulate nucleotide binding properties in SRP GTPases. A reorganization of the other G-loops and the insertion binding domain (IBD) is observed only upon transition from a diphosphate to a triphosphate nucleotide. The role of a magnesium ion during the GDP and GTP-bound states has also been observed. The binding of magnesium in the nucleotide site causes the reorientation of the ß- and γ- phosphate groups toward the jaws of the P-loop and stabilizes the binding of the nucleotide, creating a network of hydrogen and water-bridge interactions.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Cristalografia por Raios X , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Glicina/química , Glicina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Magnésio/metabolismo , Modelos Moleculares , Nucleotídeos/metabolismo , Fosfatos/química , Conformação Proteica , Domínios Proteicos , Partícula de Reconhecimento de Sinal/química , Valina/química , Valina/metabolismo
10.
Biochem J ; 465(2): 337-46, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25367669

RESUMO

Canonical single-stranded DNA-binding proteins (SSBs) from the oligosaccharide/oligonucleotide-binding (OB) domain family are present in all known organisms and are critical for DNA replication, recombination and repair. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has a 'simple' domain organization consisting of a single DNA-binding OB fold coupled to a flexible C-terminal tail, in contrast with other SSBs in this family that incorporate up to four OB domains. Despite the large differences in the domain organization within the SSB family, the structure of the OB domain is remarkably similar all cellular life forms. However, there are significant differences in the molecular mechanism of ssDNA binding. We have determined the structure of the SsoSSB OB domain bound to ssDNA by NMR spectroscopy. We reveal that ssDNA recognition is modulated by base-stacking of three key aromatic residues, in contrast with the OB domains of human RPA and the recently discovered human homologue of SsoSSB, hSSB1. We also demonstrate that SsoSSB binds ssDNA with a footprint of five bases and with a defined binding polarity. These data elucidate the structural basis of DNA binding and shed light on the molecular mechanism by which these 'simple' SSBs interact with ssDNA.


Assuntos
Proteínas Arqueais/química , DNA Arqueal/química , Proteínas de Ligação a DNA/química , Sulfolobus solfataricus/química , Proteínas Arqueais/genética , DNA Arqueal/genética , Proteínas de Ligação a DNA/genética , Humanos , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Sulfolobus solfataricus/genética
12.
Nat Commun ; 15(1): 5235, 2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38898016

RESUMO

IS1111 and IS110 insertion sequence (IS) family members encode an unusual DEDD transposase type and exhibit specific target site selection. The IS1111 group include identifiable subterminal inverted repeats (sTIR) not found in the IS110 type1. IS in both families include a noncoding region (NCR) of significant length and, as each individual IS or group of closely related IS selects a different site, we had previously proposed that an NCR-derived RNA was involved in target selection2. Here, we find that the NCR is usually downstream of the transposase gene in IS1111 family IS and upstream in the IS110 type. Four IS1111 and one IS110 family members that target different sequences are used to demonstrate that the NCR determines a short seeker RNA (seekRNA) that co-purified with the transposase. The seekRNA is essential for transposition of the IS or a cargo flanked by IS ends from and to the preferred target. Short sequences matching both top and bottom strands of the target are present in the seekRNA but their order in IS1111 and IS110 family IS is reversed. Reprogramming the seekRNA and donor flank to target a different site is demonstrated, indicating future biotechnological potential for these systems.


Assuntos
Elementos de DNA Transponíveis , Transposases , Transposases/metabolismo , Transposases/genética , Elementos de DNA Transponíveis/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Sequência de Bases
13.
Front Mol Biosci ; 8: 679584, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34113652

RESUMO

The signal recognition particle (SRP) is a ribonucleoprotein complex fundamental for co-translational delivery of proteins to their proper membrane localization and secretory pathways. Literature of the past two decades has suggested new roles for individual SRP components, 7SL RNA and proteins SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72, outside the SRP cycle. These noncanonical functions interconnect SRP with a multitude of cellular and molecular pathways, including virus-host interactions, stress response, transcriptional regulation and modulation of apoptosis in autoimmune diseases. Uncovered novel properties of the SRP components present a new perspective for the mammalian SRP as a biological modulator of multiple cellular processes. As a consequence of these findings, SRP components have been correlated with a growing list of diseases, such as cancer progression, myopathies and bone marrow genetic diseases, suggesting a potential for development of SRP-target therapies of each individual component. For the first time, here we present the current knowledge on the SRP noncanonical functions and raise the need of a deeper understanding of the molecular interactions between SRP and accessory cellular components. We examine diseases associated with SRP components and discuss the development and feasibility of therapeutics targeting individual SRP noncanonical functions.

14.
RNA Biol ; 6(4): 479-87, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19667754

RESUMO

Bacillus cereus 14579 encodes two tRNAs with the CCA anticodon, tRNA(Trp) and tRNA(Other). tRNA(Trp) was separately aminoacylated by two enzymes, TrpRS1 and TrpRS2, which share only 34% similarity and display different catalytic capacities and specificities. TrpRS1 was 18-fold more proficient at aminoacylating tRNA(Trp) with Trp, while TrpRS2 more efficiently utilizes the Trp analog 5-hydroxy Trp. tRNA(Other) was not aminoacylated by either TrpRS but instead by the combined activity of LysRS1 and LysRS2, which recognized sequence elements absent from tRNA(Trp). Polysomes were found to contain tRNA(Trp), consistent with its role in translation, but not tRNA(Other) suggesting a function outside protein synthesis. Regulation of the genes encoding TrpRS1 and TrpRS2 (trpS1 and trpS2) is dependent on riboswitch-mediated recognition of the CCA anticodon, and the role of tRNA(Other) in this process was investigated. Deletion of tRNA(Other) led to up to a 50 fold drop in trpS1 expression, which resulted in the loss of differential regulation of the trpS1 and trpS2 genes in stationary phase. These findings reveal that sequence-specific interactions with a tRNA anticodon can be confined to processes outside translation, suggesting a means by which such RNAs may evolve non-coding functions.


Assuntos
Anticódon/genética , Bacillus cereus/genética , Biossíntese de Proteínas/genética , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Aminoacilação de RNA de Transferência/genética
15.
FEMS Microbiol Lett ; 366(3)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30753435

RESUMO

The insertion sequence IS26 plays a major role in the mobilization, expression and dissemination of antibiotic resistance genes in Gram-negative bacteria. Though IS26 is abundant in sequenced genomes and in plasmids that harbour antibiotic resistance genes, only a few minor variations in the IS26 sequence have been recorded. The most common variant, IS26* (also known as IS15Δ1), encodes a Tnp26 transposase with a single amino acid substitution, G184N in the catalytic domain. Using computational modelling, this substitution was predicted to increase the length of the helix that includes the E173 residue of the catalytic DDE triad, and its effect on activity was tested. An IS26 mutant generated in vitro producing Tnp26-G184N formed cointegrates in a standard untargeted reaction at 5-fold higher frequency than IS26 producing Tnp26. When the target included a single copy of IS26, the G184N substitution increased the cointegration frequency 10-fold and the reaction was targeted and conservative. Hence, the substitution increased Tnp26 activity. The longer helix may stabilise the position of the E173 of the DDE for the catalysis reaction and the specific G184N substitution may also enhance activity by increasing binding to the terminal inverted repeats.


Assuntos
Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana/genética , Variação Genética , Bactérias Gram-Negativas/genética , Modelos Moleculares , Substituição de Aminoácidos/genética , Ativação Enzimática/genética , Bactérias Gram-Negativas/enzimologia , Ligação Proteica/genética , Transposases/genética , Transposases/metabolismo
16.
Front Mol Biosci ; 5: 7, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29459899

RESUMO

The structural flexibility of RNA allows it to exist in several shapes and sizes. Thus, RNA is functionally diverse and is known to be involved in processes such as catalysis, ligand binding, and most importantly, protein recognition. RNA can adopt different structures, which can often dictate its functionality. When RNA binds onto protein to form a ribonucleoprotein complex (RNP), multiple interactions and conformational changes occur with the RNA and protein. However, there is the question of whether there is a specific pattern for these changes to occur upon recognition. In particular when RNP complexity increases with the addition of multiple proteins/RNA, it becomes difficult to structurally characterize the overall changes using the current structural determination techniques. Hence, there is a need to use a combination of biochemical, structural and computational modeling to achieve a better understanding of the processes that RNPs are involved. Nevertheless, there are well-characterized systems that are evolutionarily conserved [such as the signal recognition particle (SRP)] that give us important information on the structural changes of RNA and protein upon complex formation.

17.
PLoS One ; 13(7): e0200387, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30044812

RESUMO

Given the increasing incidence of antibiotic resistance, antibiotics that employ new strategies are urgently needed. Bacterial survival is dependent on proper function of the signal recognition particle (SRP) and its receptor (FtsY). A unique set of interactions in FtsY:SRP-RNA represents a promising candidate for new antibiotic development as no antibiotic targets this complex and these interactions are functionally replaced by protein:protein interactions in eukaryotes. We used a Fragment Based Drug Design (FBDD) approach to search for new compounds that can bind FtsY, and have identified three lead fragments. In vitro and in vivo analyses have shown that despite a high micromolar binding affinity, one fragment has some antimicrobial properties. X-ray structures of E. coli FtsY:fragments reveal the fragments bind in the targeted RNA interaction site. Our results show that FBDD is a suitable approach for targeting FtsY:SRP-RNA for antibiotic development and opens the possibility of targeting protein:RNA interactions in general.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Acinetobacter baumannii , Sítios de Ligação , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/fisiologia , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Ressonância Magnética Nuclear Biomolecular , Espectroscopia de Prótons por Ressonância Magnética , Ressonância de Plasmônio de Superfície
18.
Curr Protein Pept Sci ; 16(8): 727-34, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25961399

RESUMO

Cell viability is only possible due to a dynamic range of essential nucleic acid-protein complex formation. DNA replication and repair, gene expression, transcription and protein synthesis are well-known processes mediated by nucleic acids (DNA and RNA) - protein interactions. Novel nucleic acid- protein complexes have been identified in the past few years aided by the development of numerous new techniques such as RNA capture or Tandem RNA Affinity Purification (TRAP). However, the biophysical and biochemical details of these interactions are mostly unknown. Here, we present three techniques (Electrophoretic Mobility Shift Assays, Microscale Thermophoresis and Surface Plasmon Resonance) that are commonly used to quantify and characterize DNA-protein and RNA-protein interactions and discuss their main advantages and limitations.


Assuntos
Fenômenos Biofísicos , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Ácidos Nucleicos/metabolismo , Termometria/métodos , Humanos , Ligação Proteica , RNA/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Ressonância de Plasmônio de Superfície
19.
FEBS Lett ; 588(20): 3649-64, 2014 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-25150170

RESUMO

Gene expression is controlled through a complex interplay among mRNAs, non-coding RNAs and RNA-binding proteins (RBPs), which all assemble along with other RNA-associated factors in dynamic and functional ribonucleoprotein complexes (RNPs). To date, our understanding of RBPs is largely limited to proteins with known or predicted RNA-binding domains. However, various methods have been recently developed to capture an RNA of interest and comprehensively identify its associated RBPs. In this review, we discuss the RNA-affinity purification methods followed by mass spectrometry analysis (AP-MS); RBP screening within protein libraries and computational methods that can be used to study the RNA-binding proteome (RBPome).


Assuntos
Proteoma/química , Proteômica/métodos , Proteínas de Ligação a RNA/metabolismo , Animais , Humanos , Proteoma/metabolismo , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/química
20.
PLoS One ; 7(7): e41248, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22815980

RESUMO

Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a pathogenicity island flanked by a predicted pseudo-tRNA, tRNA(Other), which does not function in translation. Deletion of tRNA(Other) led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNA(Other). This suggested that tRNA(Other) might be expressed as part of a larger RNA, and RACE analysis subsequently confirmed the existence of several RNA species that significantly extend both the 3' and 5'-ends of tRNA(Other). tRNA(Other) expression levels were found to be responsive to changes in extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) binding sites in the 5' leader region of one of these larger RNAs. Taken together with previous data, this study now suggests that tRNA(Other) may function by providing a tRNA-like structural element within a larger regulatory RNA. These findings illustrate that while integration of genomic islands often leaves tRNA genes intact and functional, in other instances inactivation may generate tRNA-like elements that are then recruited to other functions in the cell.


Assuntos
Bacillus cereus/efeitos dos fármacos , Bacillus cereus/genética , Resistência Microbiana a Medicamentos/genética , RNA de Transferência/genética , Biofilmes , DNA Complementar/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genoma Bacteriano , Ilhas Genômicas , Ferro/metabolismo , Óxido Nítrico/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Fenótipo , RNA de Transferência/metabolismo , Transcrição Gênica
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