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1.
EMBO Rep ; 20(12): e47964, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31680439

RESUMO

RNA-binding proteins (RBPs) participate in all steps of gene expression, underscoring their potential as regulators of RNA homeostasis. We structurally and functionally characterize Mip6, a four-RNA recognition motif (RRM)-containing RBP, as a functional and physical interactor of the export factor Mex67. Mip6-RRM4 directly interacts with the ubiquitin-associated (UBA) domain of Mex67 through a loop containing tryptophan 442. Mip6 shuttles between the nucleus and the cytoplasm in a Mex67-dependent manner and concentrates in cytoplasmic foci under stress. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation experiments show preferential binding of Mip6 to mRNAs regulated by the stress-response Msn2/4 transcription factors. Consistent with this binding, MIP6 deletion affects their export and expression levels. Additionally, Mip6 interacts physically and/or functionally with proteins with a role in mRNA metabolism and transcription such as Rrp6, Xrn1, Sgf73, and Rpb1. These results reveal a novel role for Mip6 in the homeostasis of Msn2/4-dependent transcripts through its direct interaction with the Mex67 UBA domain.

2.
Nucleic Acids Res ; 45(17): 10293-10305, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28973465

RESUMO

Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events. We determined the structures of Nrd1 RNA binding domain and its complexes with three GUAA-containing RNAs, characterized RNA binding energetics and tested rationally designed mutants in vivo. The Nrd1 structure shows an RRM domain fused with a second α/ß domain that we name split domain (SD), because it is formed by two non-consecutive segments at each side of the RRM. The GUAA interacts with both domains and with a pocket of water molecules, trapped between the two stacking adenines and the SD. Comprehensive binding studies demonstrate for the first time that Nrd1 has a slight preference for GUAA over GUAG and genetic and functional studies suggest that Nrd1 RNA binding domain might play further roles in non-coding RNAs transcription termination.


Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Terminação da Transcrição Genética , Sequência de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
3.
Nucleic Acids Res ; 44(1): 437-48, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26602689

RESUMO

Metazoan SR and SR-like proteins are important regulatory factors in RNA splicing, export, translation and RNA decay. We determined the NMR structures and nucleic acid interaction modes of Gbp2 and Hrb1, two paralogous budding yeast proteins with similarities to mammalian SR proteins. Gbp2 RRM1 and RRM2 recognise preferentially RNAs containing the core motif GGUG. Sequence selectivity resides in a non-canonical interface in RRM2 that is highly related to the SRSF1 pseudoRRM. The atypical Gbp2/Hrb1 C-terminal RRM domains (RRM3) do not interact with RNA/DNA, likely because of their novel N-terminal extensions that block the canonical RNA binding interface. Instead, we discovered that RRM3 is crucial for interaction with the THO/TREX complex and identified key residues essential for this interaction. Moreover, Gbp2 interacts genetically with Tho2 as the double deletion shows a synthetic phenotype and preventing Gbp2 interaction with the THO/TREX complex partly supresses gene expression defect associated with inactivation of the latter complex. These findings provide structural and functional insights into the contribution of SR-like proteins in the post-transcriptional control of gene expression.


Assuntos
Complexos Multiproteicos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , DNA/metabolismo , Modelos Moleculares , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/metabolismo , Ligação Proteica , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Telômero/genética , Telômero/metabolismo
4.
Structure ; 21(10): 1800-11, 2013 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-23994011

RESUMO

The seven C-terminal CCCH-type zinc fingers of Nab2p bind the poly(A) tail of mRNA (∼A25). Using NMR, we demonstrated that the first four (Zf1-Zf4) contain two structurally independent tandems (TZF12 and TZF34) and bind A12 with moderate affinity (KD = 2.3 µM). Nab2p TZF12 contains a long α helix that contacts the zinc fingers Zf1 and Zf2 to arrange them similarly to Zf6-7 in the Nab2p Zf5-7 structure. Nab2p TZF34 exhibits a distinctive two-fold symmetry of the zinc centers with mutual recognition of histidine ligands. Our mutagenesis and NMR data demonstrate that the α helix of TZF12 and Zf3 of TZF34 define the RNA-binding interface, while Zf1, Zf2, and Zf4 seem to be excluded. These results further our understanding of polyadenosine RNA recognition by the CCCH domain of Nab2p. Moreover, we describe a hypothetical mechanism for controlling poly(A) tail length with specific roles for TZF12, TZF34, and Zf5-7 domains.


Assuntos
Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Sequência Conservada , Complexos de Coordenação/química , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas de Transporte Nucleocitoplasmático/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia Estrutural de Proteína , Termodinâmica , Dedos de Zinco
5.
PLoS One ; 6(9): e24481, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21931728

RESUMO

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to ß-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1-402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role.


Assuntos
Carbono-Nitrogênio Ligases/química , Fator de Iniciação 4G em Eucariotos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas Fúngicas/química , Regulação Fúngica da Expressão Gênica , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Solubilidade
6.
J Biol Chem ; 282(3): 2101-15, 2007 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-17116658

RESUMO

Yeast Rna15 and its vertebrate orthologue CstF-64 play critical roles in mRNA 3 '-end processing and in transcription termination downstream of poly(A) sites. These proteins contain N-terminal domains that recognize the poly(A) site, but little is known about their highly conserved C-terminal regions. Here we show by NMR that the C-terminal domains of CstF-64 and Rna15 fold into a three-helix bundle with an uncommon topological arrangement. The structure defines a cluster of evolutionary conserved yet exposed residues we show to be essential for the interaction between Pcf11 and Rna15. Furthermore, we demonstrate that this interaction is critical for the function of Rna15 in 3 '-end processing but dispensable for transcription termination. The C-terminal domain of the Rna15 homologue Pti1 contains critical sequence alterations within this region that are predicted to prevent Pcf11 interaction, providing an explanation for the distinct functions of these two closely related proteins in the 3 '-end formation of RNA polymerase II transcripts. These results define the role of the C-terminal half of Rna15 and provide insight into the network of protein/protein interactions responsible for assembly of the 3 '-end processing apparatus.


Assuntos
Fator Estimulador de Clivagem/química , Processamento de Terminações 3' de RNA , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Fatores de Poliadenilação e Clivagem de mRNA/química , Sequência de Aminoácidos , Animais , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
7.
EMBO J ; 25(13): 3167-78, 2006 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-16794580

RESUMO

The recognition of specific signals encoded within the 3'-untranslated region of the newly transcribed mRNA triggers the assembly of a multiprotein machine that modifies its 3'-end. Hrp1 recognises one of such signals, the so-called polyadenylation enhancement element (PEE), promoting the recruitment of other polyadenylation factors in yeast. The molecular bases of this interaction are revealed here by the solution structure of a complex between Hrp1 and an oligonucleotide mimicking the PEE. Six consecutive bases (AUAUAU) are specifically recognised by two RNA-binding domains arranged in tandem. Both protein and RNA undergo significant conformational changes upon complex formation with a concomitant large surface burial of RNA bases. Key aspects of RNA specificity can be explained by the presence of intermolecular aromatic-aromatic contacts and hydrogen bonds. Altogether, the Hrp1-PEE structure represents one of the first steps towards understanding of the assembly of the cleavage and polyadenylation machinery at the atomic level.


Assuntos
Regiões 3' não Traduzidas , Modelos Moleculares , RNA Mensageiro/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Poliadenilação e Clivagem de mRNA/química , Adenina/química , Sequência de Aminoácidos , Ligações de Hidrogênio , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Poliadenilação , Ligação Proteica , Ribose/química , Proteínas de Saccharomyces cerevisiae/genética , Uracila/química , Fatores de Poliadenilação e Clivagem de mRNA/genética
8.
J Am Chem Soc ; 128(1): 100-16, 2006 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-16390137

RESUMO

The emergence of bacterial resistance to the major classes of antibiotics has become a serious problem over recent years. For aminoglycosides, the major biochemical mechanism for bacterial resistance is the enzymatic modification of the drug. Interestingly, in several cases, the oligosaccharide conformation recognized by the ribosomic RNA and the enzymes responsible for the antibiotic inactivation is remarkably different. This observation suggests a possible structure-based chemical strategy to overcome bacterial resistance; in principle, it should be possible to design a conformationally locked oligosaccharide that still retains antibiotic activity but that is not susceptible to enzymatic inactivation. To explore the scope and limitations of this strategy, we have synthesized several aminoglycoside derivatives locked in the ribosome-bound "bioactive" conformation. The effect of the structural preorganization on RNA binding, together with its influence on the aminoglycoside inactivation by several enzymes involved in bacterial resistance, has been studied. Our results indicate that the conformational constraint has a modest effect on their interaction with ribosomal RNA. In contrast, it may display a large impact on their enzymatic inactivation. Thus, the work presented herein provides a key example of how the conformational differences exhibited by these ligands within the binding pockets of the ribosome and of those enzymes involved in bacterial resistance can, in favorable cases, be exploited for designing new antibiotic derivatives with improved activity in resistant strains.


Assuntos
Aminoglicosídeos/química , Aminoglicosídeos/farmacologia , Acetiltransferases/química , Acetiltransferases/metabolismo , Aminoglicosídeos/síntese química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Configuração de Carboidratos , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Framicetina/análogos & derivados , Framicetina/síntese química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA/química , RNA/metabolismo , Ribose/química , Relação Estrutura-Atividade
9.
J Mol Biol ; 347(4): 719-33, 2005 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-15769465

RESUMO

The N-terminal domain of the 64 kDa subunit of the cleavage stimulation factor (CstF-64) recognizes GU-rich elements within the 3'-untranslated region of eukaryotic mRNAs. This interaction is essential for mRNA 3' end processing and transcription termination, and its strength affects the efficiency of utilization of different polyadenylation sites. The structure of the RNA-binding N-terminal domain of CstF-64 showed how the N-terminal RNA recognition motif of CstF-64 recognizes GU-rich RNAs. However, it is still perplexing how this protein can bind selectively to RNAs that are rich in G and U residues regardless of their detailed sequence composition, yet discriminate effectively against non-GU-RNAs. We investigated by NMR the dynamics of the CstF-64 RNA-binding domain, both free and bound to two GU-rich RNA sequences that represent polyadenylation regulatory elements. While the free protein displays the motional properties typical of a well-folded protein domain and is uniformly rigid, the protein-RNA interface acquires significant mobility on the micro- to millisecond time-scale once GU-rich RNAs binds to it. These motional features, we propose, are intrinsic to the functional requirement to bind all GU-rich sequences and yet to discriminate against non-GU-rich RNAs. This behavior may be a general mechanism by which some RNA-binding proteins are able to bind to classes of sequences, as opposed to a well-defined sequence or consensus.


Assuntos
Guanosina/metabolismo , Poliadenilação/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/genética , RNA/metabolismo , Uridina/metabolismo , Guanosina/genética , Humanos , Modelos Moleculares , Movimento (Física) , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , Proteínas de Ligação a RNA/genética , Especificidade por Substrato , Uridina/genética
10.
J Biol Chem ; 279(33): 34963-70, 2004 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-15178680

RESUMO

The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.


Assuntos
Proteínas Sanguíneas/química , Espectroscopia de Ressonância Magnética/métodos , Chaperonas Moleculares/química , Alanina/química , Sítios de Ligação , Clonagem Molecular , DNA/química , DNA Complementar/metabolismo , Dimerização , Biblioteca Gênica , Humanos , Ligantes , Modelos Moleculares , Chaperonas Moleculares/sangue , Mutação , Plasmídeos/metabolismo , Mutação Puntual , Prolina/química , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Tirosina/química
11.
Biochemistry ; 42(45): 13122-33, 2003 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-14609322

RESUMO

The cytotoxic ribonuclease alpha-sarcin is the best characterized member of the ribotoxin family. Ribotoxins share a common structural core, catalytic residues, and active site topology with members of the broader family of nontoxic microbial extracellular RNases. They are, however, much more specific in their biological action. To shed light on the highly specific alpha-sarcin activity, we have evaluated the structural and electrostatic interactions of its charged groups, by combining the structural and pK(a) characterization by NMR of several variants with theoretical calculations based on the Tanford-Kirkwood and Poisson-Boltzmann models. The NMR data reveal that the global conformation of wild-type alpha-sarcin is preserved in the H50Q, E96Q, H137Q, and H50/137Q variants, and that His137 is involved in an H-bond that is crucial in maintaining the active site structure and in reinforcing the stability of the enzyme. The loss of this H-bond in the H137Q and H50/137Q variants modifies the local structure of the active site. The pK(a) values of active site groups H50, E96, and H137 in the four variants have been determined by two-dimensional NMR. The catalytic dyad of E96 and H137 is not sensitive to charge replacements, since their pK(a) values vary less than +/-0.3 pH unit with respect to those of the wild type. On the contrary, the pK(a) of His50 undergoes drastic changes when compared to its value in the intact protein. These amount to an increase of 0.5 pH unit or a decrease of 1.1 pH units depending on whether a positive or negative charge is substituted at the active site. The main determinants of the pK(a) values of most of the charged groups in alpha-sarcin have been established by considering the NMR results in conjunction with those derived from theoretical pK(a) calculations. With regard to the active site residues, the H50 pK(a) is chiefly influenced by electrostatic interactions with E96 and H137, whereas the effect of the low dielectric constant and the interaction with R121 appear to be the main determinants of the altered pK(a) value of E96 and H137. Charge-charge interactions and an increased level of burial perturb the pK(a) values of the active site residues of alpha-sarcin, which can account for its reduced ribonucleolytic activity and its high specificity.


Assuntos
Domínio Catalítico/genética , Endorribonucleases/química , Endorribonucleases/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Mutagênese Sítio-Dirigida , Substituição de Aminoácidos/genética , Aspergillus/enzimologia , Aspergillus/genética , Sítios de Ligação/genética , Ácido Glutâmico/genética , Glutamina/genética , Histidina/genética , Concentração de Íons de Hidrogênio , Modelos Químicos , Ressonância Magnética Nuclear Biomolecular/métodos , Prótons , Eletricidade Estática
12.
EMBO J ; 22(11): 2821-30, 2003 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-12773396

RESUMO

Vertebrate polyadenylation sites are identified by the AAUAAA signal and by GU-rich sequences downstream of the cleavage site. These are recognized by a heterotrimeric protein complex (CstF) through its 64 kDa subunit (CstF-64); the strength of this interaction affects the efficiency of poly(A) site utilization. We present the structure of the RNA-binding domain of CstF-64 containing an RNA recognition motif (RRM) augmented by N- and C-terminal helices. The C-terminal helix unfolds upon RNA binding and extends into the hinge domain where interactions with factors responsible for assembly of the polyadenylation complex occur. We propose that this conformational change initiates assembly. Consecutive Us are required for a strong CstF-GU interaction and we show how UU dinucleotides are recognized. Contacts outside the UU pocket fine tune the protein-RNA interaction and provide different affinities for distinct GU-rich elements. The protein-RNA interface remains mobile, most likely a requirement to bind many GU-rich sequences and yet discriminate against other RNAs. The structural distinction between sequences that form stable and unstable complexes provides an operational distinction between weakly and strongly processed poly(A) sites.


Assuntos
RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Sítios de Ligação/genética , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Homologia de Sequência de Aminoácidos
13.
Protein Sci ; 12(1): 161-9, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12493839

RESUMO

Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as alpha-sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of alpha-sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild-type alpha-sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of alpha-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of alpha-sarcin on ribosomes.


Assuntos
Endorribonucleases/metabolismo , Proteínas Fúngicas , Leucina/metabolismo , Ribossomos/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Endorribonucleases/química , Endorribonucleases/genética , Endorribonucleases/farmacologia , Fungos/enzimologia , Fungos/genética , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Leucina/química , Leucina/genética , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Estabilidade de RNA , RNA Ribossômico/metabolismo , Rabdomiossarcoma/metabolismo , Ribossomos/efeitos dos fármacos , Células Tumorais Cultivadas
14.
J Biomol NMR ; 24(4): 301-16, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12522295

RESUMO

The cytotoxic ribonuclease alpha-sarcin is a 150-residue protein that inactivates ribosomes by selectively cleaving a single phosphodiester bond in a strictly conserved rRNA loop. In order to gain insights on the molecular basis of its highly specific activity, we have previously determined its solution structure and studied its electrostatics properties. Here, we complement those studies by analysing the backbone dynamics of alpha-sarcin through measurement of longitudinal relaxation rates R1, off resonance rotating frame relaxation rates R1 rho, and the 15N[1H] NOE of the backbone amide 15N nuclei at two different magnetic field strengths (11.7 and 17.6 T). The two sets of relaxation parameters have been analysed in terms of the reduced spectral density mapping formalism, as well as by the model-free approach. alpha-Sarcin behaves as an axial symmetric rotor of the prolate type (D(axially)/D(radially)=1.16 +/- 0.02) which tumbles with a correlation time tau(m) of 7.54 +/- 0.02 ns. The rotational diffusion properties have been also independently evaluated by hydrodynamic calculations and are in good agreement with the experimental results. The analysis of the internal dynamics reveals that alpha-sarcin is composed of a rigid hydrophobic core and some exposed segments which undergo fast (ps to ns) internal motions. Slower motions in the mu s to ms time scale are less abundant and in some cases can be assigned to specific motional processes. All dynamic data are discussed in relation to the role of some particular residues of alpha-sarcin in the process of recognition of its ribosomal target.


Assuntos
Endorribonucleases/química , Proteínas Fúngicas , Ressonância Magnética Nuclear Biomolecular/métodos , Difusão , Magnetismo , Modelos Moleculares , Movimento (Física) , Isótopos de Nitrogênio , Maleabilidade , Estrutura Secundária de Proteína , Rotação
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