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1.
Proteins ; 79(8): 2566-77, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21633970

RESUMO

GCN5-related N-acetyltransferases (GNATs) are the most widely distributed acetyltransferase systems among all three domains of life. GNATs appear to be involved in several key processes, including microbial antibiotic resistance, compacting eukaryotic DNA, controlling gene expression, and protein synthesis. Here, we report the crystal structure of a putative GNAT Ta0374 from Thermoplasma acidophilum, a hyperacidophilic bacterium, that has been determined in an apo-form, in complex with its natural ligand (acetyl coenzyme A), and in complex with a product of reaction (coenzyme A) obtained by cocrystallization with spermidine. Sequence and structural analysis reveals that Ta0374 belongs to a novel protein family, PaiA, involved in the negative control of sporulation and degradative enzyme production. The crystal structure of Ta0374 confirms that it binds acetyl coenzyme A in a way similar to other GNATs and is capable of acetylating spermidine. Based on structural and docking analysis, it is expected that Glu53 and Tyr93 are key residues for recognizing spermidine. Additionally, we find that the purification His-Tag in the apo-form structure of Ta0374 prevents binding of acetyl coenzyme A in the crystal, though not in solution, and affects a chain-flip rotation of "motif A" which is the most conserved sequence among canonical acetyltransferases.


Assuntos
Acetiltransferases/química , Proteínas Arqueais/química , Cristalografia por Raios X/métodos , Thermoplasma/enzimologia , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos
2.
Int J Tuberc Lung Dis ; 13(4): 494-9, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19335956

RESUMO

OBJECTIVE: To analyse the extent of drug resistance in clinical isolates of Mycobacterium tuberculosis from patients attending various tuberculosis (TB) clinics in Kerala, India. DESIGN: Mycobacteria were isolated from sputum samples of TB patients. Isolates from 92 new and 104 retreatment cases were tested for resistance to four first-line drugs (isoniazid, rifampicin, ethambutol and streptomycin). RESULTS: Twenty-three per cent of the isolates from new cases and 14% from retreatment cases were pan-susceptible, and the rest were resistant to at least one of the drugs. Multidrug-resistant isolates accounted for 5.4% among new cases and 16.4% among retreatment cases. It should be noted that 18.5% of the isolates were mycobacteria other than tuberculosis. CONCLUSION: There is an urgent need for statewide surveys to assess the level of drug resistance using quality-assured culture and drug susceptibility services. Considering that the Revised National TB Control Programme in India has been made operational nationwide, this kind of screening should be made mandatory under the programme to effectively control the spread of TB.


Assuntos
Farmacorresistência Bacteriana Múltipla , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/farmacologia , Humanos , Índia , Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia
3.
Mol Cell Biol ; 19(8): 5601-7, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10409749

RESUMO

We have examined structural differences between the proto-oncogene c-Myb and the cyclic AMP-responsive factor CREB that underlie their constitutive or signal-dependent activation properties. Both proteins stimulate gene expression via activating regions that articulate with a shallow hydrophobic groove in the KIX domain of the coactivator CREB-binding protein (CBP). Three hydrophobic residues in c-Myb that are conserved in CREB function importantly in cellular gene activation and in complex formation with KIX. These hydrophobic residues are assembled on one face of an amphipathic helix in both proteins, and mutations that disrupt c-Myb or CREB helicity in this region block interaction of either factor with KIX. Binding of the helical c-Myb domain to KIX is accompanied by a substantial increase in entropy that compensates for the comparatively low enthalpy of complex formation. By contrast, binding of CREB to KIX entails a large entropy cost due to a random coil-to-helix transition in CREB that accompanies complex formation. These results indicate that the constitutive and inducible activation properties of c-Myb and CREB reflect secondary structural characteristics of their corresponding activating regions that influence the thermodynamics of formation of a complex with CBP.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Regulação da Expressão Gênica , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas/química , Transativadores/química , Sequência de Aminoácidos , Animais , Proteína de Ligação a CREB , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-myb , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico , Transdução de Sinais , Relação Estrutura-Atividade , Termodinâmica , Transativadores/metabolismo , Transativadores/fisiologia , Ativação Transcricional , Transfecção
4.
Structure ; 2(1): 17-32, 1994 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-8075980

RESUMO

BACKGROUND: Under certain conditions, homopyrimidine oligonucleotides can bind to complementary homopurine sequences in homopurine-homopyrimidine segments of duplex DNA to form triple helical structures. Besides having biological implications in vivo, this property has been exploited in molecular biology applications. This approach is limited by a lack of knowledge about the recognition by the third strand of pyrimidine residues in Watson-Crick base pairs. RESULTS: We have therefore determined the solution structure of a pyrimidine.purine.pyrimidine (Y.RY) DNA triple helix containing a guanine residue in the third strand which was postulated to specifically recognize a thymine residue in a Watson-Crick TA base pair. The structure was solved by combining NMR-derived restraints with molecular dynamics simulations conducted in the presence of explicit solvent and counter ions. The guanine of the G-TA triple is tilted out of the plane of its target TA base pair towards the 3'-direction, to avoid a steric clash with the thymine methyl group. This allows the guanine amino protons to participate in hydrogen bonds with separate carbonyls, forming one strong bond within the G-TA triple and a weak bond to an adjacent T.AT triple. Dramatic variations in helical twist around the guanine residue lead to a novel stacking interaction. At the global level, the Y.RY DNA triplex shares several structural features with the recently solved solution structure of the R.RY DNA triplex. CONCLUSIONS: The formation of a G.TA triple within an otherwise pyrimidine.purine.pyrimidine DNA triplex causes conformational realignments in and around the G.TA triple. These highlight new aspects of molecular recognition that could be useful in triplex-based approaches to inhibition of gene expression and site-specific cleavage of genomic DNA.


Assuntos
DNA/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Composição de Bases , Sequência de Bases , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/síntese química , Purinas , Pirimidinas , Soluções , Estresse Mecânico
5.
Structure ; 2(5): 395-405, 1994 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-8081755

RESUMO

BACKGROUND: DNA triplexes are higher-order nucleic acid structures with potential roles in gene regulation and hence biochemical and therapeutic applications. The stabilizing influence exerted by water molecules on the conformation of the DNA duplex is well known. However, the role of water molecules in the DNA triple helix has not been investigated. We have previously determined the solution structures of the purine.purine.pyrimidine (R.RY) and pyrimidine.purine.pyrimidine (Y.RY) structural motifs in DNA triplexes and identified both the global helical parameters, as well as local helical distortions associated with non-standard base triple pairing alignments. RESULTS: Here we have used homonuclear two-dimensional NMR spectroscopy to define the hydration sites in R.RY and Y.RY DNA triplexes in aqueous solution. Long-lived hydration sites with residence times exceeding 1 nanosecond have been identified in the new groove formed by the Hoogsteen paired strands in both triplexes. Distinctive patterns of hydration are displayed by each triplex in the remaining two grooves. CONCLUSION: The role played by water molecules in DNA triplexes appears to be similar to that played in duplexes. By binding to specific sites, particularly in the narrow groove formed by the Hoogsteen paired strands whose phosphate groups are in close proximity, water molecules may stabilize the triplex by shielding it against unfavorable electrostatic interactions.


Assuntos
DNA/química , Oligodesoxirribonucleotídeos/química , Água/química , Composição de Bases , Sequência de Bases , Temperatura Alta , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Soluções/química
6.
Structure ; 1(2): 135-52, 1993 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-8069626

RESUMO

BACKGROUND: Oligonucleotide-directed triple helix formation allows sequence specific recognition of double helical DNA. This powerful approach has been used to inhibit gene transcription in vitro and to mediate single site specific cleavage of a human chromosome. RESULTS: Using a combined NMR and molecular dynamics approach (including relaxation matrix refinement), we have determined the solution structure of an intramolecular purine.purine.pyrimidine (R.RY) DNA triplex containing guanines and thymines in the third strand to high resolution. Our studies define the G.GC and T.AT base triple pairing alignments in the R.RY triplex and identify the structural discontinuities in the third strand associated with the non-isomorphism of the base triples. The 5'-d(TpG)-3' base steps exhibit a pronounced increase in axial rise and reduction in helical twist, while the reverse is observed, to a lesser extent at 5'-d(GpT)-3' steps. A third groove is formed between the purine-rich third strand and the pyrimidine strand. It is wider and deeper than the other two grooves. CONCLUSIONS: Our structure of the R.RY DNA triplex will be important in the design of oligonucleotide probes with enhanced specificity and affinity for targeting in the genome. The third groove presents a potential target for binding additional ligands.


Assuntos
DNA/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Adenina , Composição de Bases , Sequência de Bases , Gráficos por Computador , Citosina , Guanina , Ligação de Hidrogênio , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Dados de Sequência Molecular , Purinas , Pirimidinas , Timina
7.
Structure ; 4(6): 725-33, 1996 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-8805553

RESUMO

BACKGROUND: RNA splicing is both ubiquitous and essential for the maturation of precursor mRNA molecules in eukaryotes. The process of trans-splicing involves the transfer of a short spliced leader (SL) RNA sequence to a consensus acceptor site on a separate pre-mRNA transcript. In Caenorhabditis elegans, a majority of pre-mRNA transcripts receive the 22-nucleotide SL from the SL1 RNA. Very little is known about the various roles that RNA structures play in the complex conformational rearrangements and reactions involved in premRNA splicing. RESULTS: We have determined the solution structure of a domain of the first stem loop of the SL1 RNA of C. elegans, using homonuclear and heteronuclear NMR techniques; this domain contains the splice-donor site and a nine-nucleotide hairpin loop. In solution, the SL1 RNA fragment adopts a stem-loop structure: nucleotides in the stem region form a classical A-type helix while nucleotides in the hairpin loop specify a novel conformation that includes a helix, that extends for the first three residues; a syn guanosine nucleotide at the turn region; and an extrahelical adenine that defines a pocket with nucleotides at the base of the loop. CONCLUSION: The proximity of this pocket to the splice donor site, combined with the observation that the nucleotides in this motif are conserved among all nematode SL RNAs, suggests that this pocket may provide a recognition site for a protein or RNA molecule in the trans-splicing process.


Assuntos
Caenorhabditis elegans/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA/genética , RNA/química , Animais , Sequência de Bases , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Transcrição Gênica/genética
8.
J Mol Biol ; 241(4): 600-19, 1994 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-8057381

RESUMO

The solution structure of a pyrimidine.purine.pyrimidine DNA triplex containing a novel T.CG base-triple has been determined via two and three-dimensional NMR spectroscopy and restrained molecular dynamics simulations incorporating explicit solvent and counter-ions. The T.CG triple, which expands the triplex code, is stabilized by a single hydrogen bond between the O-2 atom of thymine and the free amino proton of cytosine in the Watson-Crick C.G base-pair. This hydrogen bonding alignment produces large variations in helical twist at the dinucleotide steps involving the thymine residue. Localized structural perturbations in the purine-rich strand of the molecule are observed around the cytosine residue in the T.CG triple. Globally, the triplex resembles the solution structure of a previously solved pyrimidine.purine.pyrimidine DNA triplex containing an unusual G.TA triple. Also conserved are the sites and patterns of hydration in the two triplexes.


Assuntos
Oligodesoxirribonucleotídeos/química , Sequência de Bases , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fósforo , Prótons , Soluções , Água
9.
J Mol Biol ; 234(1): 188-97, 1993 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-8230198

RESUMO

This study reports on an NMR characterization of an intramolecular purine.purine.pyrimidine (R.RY) triplex containing a central A.AT triple in addition to G.GC and T.AT triples. Our studies establish an A(anti).AT alignment with reversed Hoogsteen pairing stabilized by two N6H ... N hydrogen bonds between adenine residues in the third strand and the purine strand of the duplex. This result, combined with our earlier demonstration of G(anti).GC and T(anti).AT pairing alignments, has provided a definitive experimental approach for differentiating between the base triple pairing possibilities proposed previously by Beal and Dervan for R.RY triplexes.


Assuntos
DNA/ultraestrutura , Conformação de Ácido Nucleico , DNA/química , Desoxirribonucleotídeos/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Purinas , Pirimidinas
10.
J Mol Biol ; 221(4): 1403-18, 1991 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-1942059

RESUMO

Recently, P.A. Beal and P.B. Dervan, expanding on earlier observations by others, have established the formation of purine.purine.pyrimidine triple helices stabilized by G.GC, A.AT and T.AT base triples where the purine-rich third strand was positioned in the major groove of the Watson-Crick duplex and anti-parallel to its purine strand. The present nuclear magnetic resonance (n.m.r.) study characterizes the base triple pairing alignments and strand direction in a 31-mer deoxyoligonucleotide that intramolecularly folds to generate a 7-mer (R/Y-)n.(R+)n(Y-)n triplex with the strands linked by two T5 loops and stabilized by potential T.AT and G.GC base triples. (R and Y stand for purine and pyrimidine, respectively, while the signs establish the strand direction.) This intramolecular triplex gives well-resolved exchangeable and non-exchangeable proton spectra with Li+ as counterion in aqueous solution. These studies establish that the T1 to C7 pyrimidine and the G8 to A14 purine strands are anti-parallel to each other and align through Watson-Crick A.T and G.C pair formation. The T15 to G21 purine-rich third strand is positioned in the major groove of this duplex and pairs through Hoogsteen alignment with the purine strand to generate T.AT and G.GC triples. Several lines of evidence establish that the thymidine and guanosine bases in the T15 to G21 purine-rich third strand adopt anti glycosidic torsion angles under conditions where this strand is aligned anti-parallel to the G8 to A14 purine strand. We have also recorded imino proton n.m.r. spectra for an (R-)n.(R+)n(Y-)n triplex stabilized by G.GC and A.AT triples through intramolecular folding of a related 31-mer deoxyoligonucleotide with Li+ as counterion. The intramolecular purine.purine.pyrimidine triplexes containing unprotonated G.GC, A.AT and T.AT triples are stable at basic pH in contrast to pyrimidine.purine.pyrimidine triplexes containing protonated C+.GC and T.AT triples, which are only stable at acidic pH.


Assuntos
Composição de Bases/fisiologia , DNA/química , Purinas/metabolismo , Pirimidinas/metabolismo , Sequência de Bases , DNA/metabolismo , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Temperatura
11.
J Mol Biol ; 252(3): 314-27, 1995 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-7563053

RESUMO

The process of trans-splicing involves the transfer of a short spliced leader (SL) RNA sequence to a consensus acceptor site on a separate pre-mRNA transcript. In this study, the first stem loop of the SL1 RNA from the nematode Caenorhabditis elegans was examined by homonuclear and heteronuclear NMR. Results of enzymatic cleavage patterns established that the first 36 nucleotides (which includes the splice site and a complementary base-paired region surrounding a nine-nucleotide hairpin loop) remain structurally independent of the rest of the 100-nucleotide full-length transcript. A comparison of exchangeable and non-exchangeable proton chemical shifts in the region of the splice site and loop between the native sequence and a modified 26-nucleotide fragment from which an asymmetric internal loop had been deleted was made. There was no significant difference between the resonance locations of the equivalent protons in the two molecules, establishing that there was no tertiary interaction between the hairpin and internal loops. Full chemical shift assignments of 1H, 13C, and 15N chemical shifts were obtained for the modified fragment by multidimensional homonuclear and heteronuclear NMR spectroscopy. The stem adopts an A-form helix typical of RNA. The A-type helical conformation of the stem appears to continue for the first three nucleotides of the 5' side of the loop, followed by a guanosine residue in a syn conformation about the glycosidic bond. Base stacking is not seen on the 3' side of the loop. There was no evidence for formation of Watson-Crick base-pairs within the loop, but several long distance NOEs indicated cross-loop contacts, indicative of a structured loop. The final loop residues, an adenine which is conserved among all known nematode SL RNA sequences, adopts an extrahelical conformation.


Assuntos
Conformação de Ácido Nucleico , Splicing de RNA , RNA de Helmintos/química , RNA Nuclear Heterogêneo/química , RNA Mensageiro/química , Animais , Composição de Bases , Sequência de Bases , Caenorhabditis , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , RNA de Helmintos/genética , RNA de Helmintos/isolamento & purificação , RNA Nuclear Heterogêneo/genética , RNA Nuclear Heterogêneo/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Terminologia como Assunto , Transcrição Gênica
12.
J Mol Biol ; 302(5): 1101-17, 2000 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-11183777

RESUMO

The hydration of a high-affinity protein-DNA complex involving the three amino terminal zinc finger domains of transcription factor IIIA (TFIIIA) and a 15-base-pair DNA duplex was investigated by NMR spectroscopy and molecular dynamics (MD) simulations. Intermolecular nuclear Overhauser effects (NOEs) between protein and water provided an experimental basis for identifying potential sites of hydration. These initial assignments were evaluated with the aid of two, 2 ns MD simulations of the protein-DNA complex conducted with the explicit inclusion of water solvent. The two independent simulations produced similar trends in terms of water residence times around the solute, and these results were used to separate protein-water NOEs from alternate exchange-relayed cross peaks. Furthermore, only six of the 170 protons which failed to show intermolecular NOEs to solvent showed nearby long-resident water molecules in the MD simulations, illustrating an impressive level of agreement between theory and experiment. Analyses of the MD trajectories also allowed an examination of the role of water in recognition and binding affinity of the zinc fingers with DNA. The interface is well hydrated, characterized by direct contacts between the protein and DNA, as well as mediating water bridges. Approximately 18 water-mediated hydrogen bonds between the protein and DNA were observed on average. Roughly half of these were water molecules with long residence times that are most likely to be important for binding, since they involve residues which have been shown through biochemical studies to be crucial for protein-DNA binding. This level of atomic detail could not otherwise be established through the existing NMR and crystal structures of the TFIIIA-DNA complex.


Assuntos
Simulação por Computador , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Água/metabolismo , Dedos de Zinco , Sítios de Ligação , DNA/química , DNA/genética , Entropia , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica , Prótons , Solventes , Fator de Transcrição TFIIIA , Água/química
13.
J Mol Biol ; 287(5): 859-65, 1999 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-10222196

RESUMO

A number of signal-dependent and development-specific transcription factors recruit CREB binding protein (CBP) for their transactivation function. The KIX domain of CBP is a common docking site for many of these transcription factors. We recently determined the solution structure of the KIX domain complexed to one of its targets, the Ser133-phosphorylated kinase inducible transactivation domain (pKID) of the cyclic AMP response element binding protein. The NMR studies have now been extended to a slightly longer KIX construct that, unlike the original KIX construct, is readily amenable to structural analysis in both the free and pKID-bound forms. This addition of six residues (KRRSRL) to the C terminus of the original construct elongates the C-terminal alpha3 helix of KIX by about eight residues. On the basis of the NMR structure of the original KIX construct, residues in the extended helix are predicted to be solvent exposed and thus are not expected to contribute to the hydrophobic core of the domain. Their role appears to be in the stabilization of the alpha3 helix through favorable electrostatic interactions with the helix dipole, which in turn confers stability on the core of the KIX domain. These results have important implications for the identification of novel protein domain boundaries. Chemical shift perturbation mapping firmly establishes a similar mode of pKID binding to the longer KIX construct and rules out any additional intermolecular interactions between residues in the C-terminal extension and pKID.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Transativadores/química , Transativadores/metabolismo , Acetiltransferases/química , Acetiltransferases/metabolismo , Sítios de Ligação , Proteína de Ligação a CREB , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência
14.
FEBS Lett ; 430(3): 317-22, 1998 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-9688563

RESUMO

Phosphorylation of Ser133 within the kinase inducible transactivation domain (KID) of the transcription factor CREB potentiates interaction with the KIX domain of coactivator CBP. Heteronuclear NMR spectroscopic analyses reveal that the KID domain is largely unstructured except for residues that comprise the alphaA helix in the pKID-KIX complex, which populate helical conformations to a significant extent (>50%). The helical content in the alphaB region is very small in the non-phosphorylated form (approximately 10%) although a small increase is detected upon Ser133 phosphorylation. The intrinsic bias towards helical conformations probably facilitates folding of the KID domain upon binding to KIX while the principal role of the phosphate group appears to be largely in mediating the intermolecular interactions in the pKID-KIX complex.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Estrutura Secundária de Proteína , Serina/química , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Proteínas Quinases , Ratos , Ativação Transcricional
15.
Biochemistry ; 31(9): 2514-23, 1992 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-1547235

RESUMO

Two- and three-dimensional homonuclear 1H NMR spectroscopic techniques have been applied to obtain nearly complete nonexchangeable proton assignments for a 31-residue intramolecular pyrimidine.purine.pyrimidine DNA triplex containing a central G.TA triple in D2O. An assignment strategy for obtaining resonance assignments for DNA protons from a 3D NOESY-TOCSY spectrum is proposed. The strategy utilizes the H1'/H5 omega 3 planes and relies on the recognition of cross-peak patterns for obtaining both intraresidue as well as sequential assignments. On the basis of the cross-peaks observed in the 2D and 3D spectra, a few structural features of the triplex have been delineated qualitatively. All three strands of the triplex adopt a right-handed helical conformation, and, despite the introduction of a central purine guanosine, there is no evidence for major structural distortions in the protonated third strand on the basis of a qualitative interpretation of NMR data. Several interstrand contacts between the purine and the Hoogsteen pyrimidine strands are observed which define the relative orientation of the bases and sugars in these two strands. The presence of strong NOEs between the methyl protons of thymine and the H1' proton of guanosine defines the preferred base-pairing alignment of guanosine at the G.TA triple site. The general approaches illustrated in this study extend the range of DNA molecules accessible for detailed structural investigation by high-resolution NMR spectroscopy.


Assuntos
Adenosina/química , DNA/química , Guanosina/química , Espectroscopia de Ressonância Magnética , Timidina/química , Sequência de Bases , Deutério , Dados de Sequência Molecular , Conformação de Ácido Nucleico
16.
Biochemistry ; 32(41): 11228-34, 1993 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-8218188

RESUMO

As a part of our ongoing efforts to define the structural aspects of unusual pairing alignments in DNA triplexes by nuclear magnetic resonance spectroscopy, we have examined the structural role of a nonnatural deoxyribonucleoside, P1, that has been shown to mediate the recognition of GC base pairs in pyrimidine-purine-pyrimidine DNA triplexes [Koh, J.S., & Dervan, P.B. (1992) J. Am. Chem Soc. 114, 1470]. A qualitative interpretation of the NMR data indicates that this analog of protonated cytosine is readily accommodated in the third strand segment of an intramolecular triplex system. Furthermore, the observed NOE patterns position the imino and amino protons of P1 opposite the N7 and O6 atoms of guanine, respectively, consistent with the previously proposed pairing scheme.


Assuntos
Composição de Bases , DNA/química , Desoxirribonucleosídeos/química , Espectroscopia de Ressonância Magnética , Sequência de Bases , Citosina , DNA/metabolismo , Desoxirribonucleosídeos/metabolismo , Guanina , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Estrutura Molecular
17.
Biochemistry ; 30(37): 9022-30, 1991 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-1654085

RESUMO

We reported previously on NMR studies of (Y+)n.(R+)n(Y-)n DNA triple helices containing one oligopurine strand (R)n and two oligopyrimidine strands (Y)n stabilized by T.AT and C+.GC base triples [de los Santos, C., Rosen, M., & Patel, D. J. (1989) Biochemistry 28, 7282-7289]. Recently, it has been established that guanosine can recognize a thymidine.adenosine base pair to form a G.TA triple in an otherwise (Y+)n.(R+)n(Y-)n triple-helix motif. [Griffin, L. C., & Dervan, P. B. (1989) Science 245, 967-971]. The present study extends the NMR research to the characterization of structural features of a 31-mer deoxyoligonucleotide that folds intramolecularly into a 7-mer (Y+)n.(R+)n(Y-)n triplex with the strands linked through two T5 loops and that contains a central G.TA triple flanked by T.AT triples. The G.TA triplex exhibits an unusually well resolved and narrow imino and amino exchangeable proton and nonexchangeable proton spectrum in H2O solution, pH 4.85, at 5 degrees C. We have assigned the imino protons of thymidine and amino protons of adenosine involved in Watson-Crick and Hoogsteen pairing in T.AT triples, as well as the guanosine imino and cytidine amino protons involved in Watson-Crick pairing and the protonated cytidine imino and amino protons involved in Hoogsteen pairing in C+.GC triples in the NOESY spectrum of the G.TA triplex. The NMR data are consistent with the proposed pairing alignment for the G.TA triple where the guanosine in an anti orientation pairs through a single hydrogen bond from one of its 2-amino protons to the 4-carbonyl group of thymidine in the Watson-Crick TA pair.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
DNA/química , Nitrogênio/química , Conformação de Ácido Nucleico , Prótons , Nucleotídeos de Purina/química , Nucleotídeos de Pirimidina/química , Composição de Bases , Sequência de Bases , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Soluções , Temperatura
18.
Nat Struct Biol ; 4(8): 605-8, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9253405

RESUMO

The three N-terminal zinc fingers of transcription factor IIIA bind in the DNA major groove. Substantial packing interfaces are formed between adjacent fingers, the linkers lose their intrinsic flexibility upon DNA binding, and several lysine side chains implicated in DNA recognition are dynamically disordered.


Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Fragmentos de Peptídeos/química , Fatores de Transcrição/química , Dedos de Zinco , Sequência de Aminoácidos , Lisina/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Fator de Transcrição TFIIIA
19.
Cell ; 91(6): 741-52, 1997 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-9413984

RESUMO

The nuclear factor CREB activates transcription of target genes in part through direct interactions with the KIX domain of the coactivator CBP in a phosphorylation-dependent manner. The solution structure of the complex formed by the phosphorylated kinase-inducible domain (pKID) of CREB with KIX reveals that pKID undergoes a coil-->helix folding transition upon binding to KIX, forming two alpha helices. The amphipathic helix alphaB of pKID interacts with a hydrophobic groove defined by helices alpha1 and alpha3 of KIX. The other pKID helix, alphaA, contacts a different face of the alpha3 helix. The phosphate group of the critical phosphoserine residue of pKID forms a hydrogen bond to the side chain of Tyr-658 of KIX. The structure provides a model for interactions between other transactivation domains and their targets.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Nucleares/metabolismo , Conformação Proteica , Transativadores , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína de Ligação a CREB , Sequência Consenso , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
20.
Mol Cell ; 2(3): 353-9, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9774973

RESUMO

Ser-133 phosphorylation of CREB within the kinase-inducible domain (KID) promotes target gene activation via complex formation with the KIX domain of the coactivator CBP. Concurrent phosphorylation of CREB at Ser-142 inhibits transcriptional induction via an unknown mechanism. Unstructured in the free state, KID folds into a helical structure upon binding to KIX. Using site-directed mutagenesis based on the NMR structure of the KID:KIX complex, we have examined the mechanisms by which Ser-133 and Ser-142 phosphorylation regulate CREB activity. Our results indicate that phospho-Ser-133 stablizes whereas phospho-Ser-142 disrupts secondary structure-mediated interactions between CREB and CBP. Thus, differential phosphorylation of CREB may form the basis by which upstream signals regulate the specificity of target gene activation.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fosfosserina , Estrutura Secundária de Proteína , Ativação Transcricional , Sequência de Aminoácidos , Caseína Quinase II , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Biblioteca de Peptídeos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina , Especificidade por Substrato , Transfecção
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