RESUMO
Metal ions are critical for RNA structure and enzymatic activity. We present the structure of an asymmetric RNA loop that binds metal ions and has an essential function in a bacteriophage packaging motor. Prohead RNA is a noncoding RNA that is required for genome packaging activity in phi29-like bacteriophage. The loops in GA1 and phi29 bacteriophage share a conserved adenine that forms a base triple, although the structural context for the base triple differs. NMR relaxation studies and femtosecond time-resolved fluorescence spectroscopy reveal the dynamic behavior of the loop in the metal ion bound and unbound forms. The mechanism of metal ion binding appears to be an induced conformational change between two dynamic ensembles rather than a conformational capture mechanism. These results provide experimental benchmarks for computational models of RNA-metal ion interactions.
Assuntos
Fagos Bacilares/genética , Metais/química , RNA Viral/química , Ligação de Hidrogênio , Íons/química , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , RNA Viral/metabolismo , Espectrometria de FluorescênciaRESUMO
Efforts toward the development of RNA-based drug leads have been challenging because of the complexity and dynamic nature of RNA structures as therapeutic targets. The transactivation response (TAR) RNA and cognate Tat protein of HIV have long been recognized as promising antiviral targets, and recent works have identified potentially potent inhibitors of the viral RNA-protein interaction. A new class of such inhibitors, conformationally constrained cyclic peptide mimetics of Tat, has been demonstrated to inhibit the HIV life cycle. We have previously probed the complexity and dynamics of TAR RNAs in their free states, as well as conformational shifting by various peptide and small molecule ligands. In this work, we have used an ultrafast dynamics approach to probe the interactions between TAR RNAs and one of the representatives of cyclic peptide inhibitors, L22. Our studies demonstrated that cyclic L22 specifically recognizes TAR RNAs with a unique single binding site compared to two binding sites for linear Tat protein. Although both Tat and L22 bind to the TAR RNAs as a ß-hairpin structure, cyclization in L22 allows it to be a more efficient ligand from a population shifting perspective. This study provided unique insights into drug design with desired properties to differentiate similar structures based on distinct dynamic behaviors.
Assuntos
Fármacos Anti-HIV/química , Desenho de Fármacos , Modelos Moleculares , Peptídeos Cíclicos/química , RNA Viral/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , HIV-1/metabolismo , HIV-2/metabolismo , Cinética , Ligantes , Conformação de Ácido Nucleico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Dobramento de RNA/efeitos dos fármacos , RNA Viral/química , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/farmacologia , Espectrometria de Fluorescência , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Noncoding 7SK snRNA is believed to play an important role in the recruitment of P-TEFb by viral protein Tat to stimulate HIV processive transcription. Because HIV-2 TAR RNA and 7SK both evolved to feature a dinucleotide bulge region, compared to the trinucleotide bulge for HIV-1 TAR, ultrafast time-resolved fluorescence spectroscopy has been used to probe the conformational landscape of HIV-2 TAR and 7SK-SL4 RNA to monitor the conformational changes upon Tat binding. Our studies demonstrate that both HIV-1/2 TAR and 7SK-SL4 sample heterogeneous ensembles in the free state and undergo distinct conformational transitions upon Tat binding. These findings provide exquisite knowledge on the conformational complexity and intricate mechanism of molecular recognition and pave the way for drug design and discovery that incorporate dynamics information.
Assuntos
Repetição Terminal Longa de HIV , HIV-1/metabolismo , HIV-2/metabolismo , RNA Nuclear Pequeno/química , RNA Viral/química , Ribonucleoproteínas Nucleares Pequenas/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Conformação de Ácido Nucleico , Ligação Proteica , RNA Viral/genéticaRESUMO
The gene 5 protein (g5p) encoded by the Ff strains of Escherichia coli bacteriophages is a dimeric single-stranded DNA-binding protein (SSB) that consists of two identical OB-fold (oligonucleotide/oligosaccharide-binding) motifs. Ultrafast time-resolved fluorescence measurements were carried out to investigate the effect of g5p binding on the conformation of 2-aminopurine (2AP) labels positioned between adenines or cytosines in the 16-nucleotide antiparallel tails of DNA hairpins. The measurements revealed significant changes in the conformational heterogeneity of the 2AP labels caused by g5p binding. The extent of the changes was dependent on sub-binding-site location, but generally resulted in base unstacking. When bound by g5p, the unstacked 2AP population increased from â¼ 22% to 59-67% in C-2AP-C segments and from 39% to 77% in an A-2AP-A segment. The OB-fold RPA70A domain of the human replication protein A also caused a significant amount of base unstacking at various locations within the DNA binding site as evidenced by steady-state fluorescence titration measurements using 2AP-labeled 5-mer DNAs. These solution studies support the concept that base unstacking at most of a protein's multiple sub-binding-site loci may be a feature that allows non-sequence specific OB-fold proteins to bind to single-stranded DNAs (ssDNAs) with minimal preference for particular sequences.
Assuntos
Proteínas de Ligação a DNA , Nucleotídeos , Sequência de Bases , DNA/metabolismo , DNA de Cadeia Simples , Proteínas de Ligação a DNA/química , Humanos , Ligação Proteica , Proteínas Virais/genéticaRESUMO
The dynamic behavior of the rRNA A-site plays an important functional role. We have employed femtosecond time-resolved spectroscopy to investigate the nature of the conformational dynamics. In the drug-free state, the A-site samples multiple distinct conformations. Drug binding shifts the population distribution in a drug-specific manner. Motions of bases on nanosecond and picosecond time scales are differentially affected by the drug binding. Our results underscore the importance of understanding the detailed dynamic picture of molecular recognition by resolving dynamics in the distinct picosecond time regime and facilitate development of antimicrobial drugs targeting dynamic RNAs.
Assuntos
RNA Ribossômico/química , Adenina/química , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Sítios de Ligação , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Polarização de Fluorescência , Cinética , Conformação de Ácido Nucleico/efeitos dos fármacos , RNA Bacteriano/química , RNA Bacteriano/efeitos dos fármacos , RNA Bacteriano/metabolismo , RNA Ribossômico/efeitos dos fármacos , RNA Ribossômico/metabolismoRESUMO
Gene 5 protein (g5p) is a dimeric single-stranded DNA-binding protein encoded by Ff strains of Escherichia coli bacteriophages. The 2-fold rotationally symmetric binding sites of a g5p dimer each bind to four nucleotides, and the dimers bind with high cooperativity to saturate antiparallel single-stranded DNA (ssDNA) strands. Ultrafast time-resolved fluorescence spectroscopies were used to investigate the conformational heterogeneity and dynamics of fluorescent 2-aminopurine (2AP) labels sequestered by bound g5p. The 2AP labels were positioned within the noncomplementary antiparallel tail sequences of d(AC)(8) or d(AC)(9) of hairpin constructs so that each fluorescent label could probe a different subsite location within the DNA-binding site of g5p. Circular dichroism and isothermal calorimetric titrations yielded binding stoichiometries of approximately six dimers per oligomer hairpin when tails were of these lengths. Mobility shift assays demonstrated the formation of a single type of g5p-saturated complex. Femtosecond time-resolved fluorescence spectroscopy showed that the 2AP in the free (non-protein-bound) DNAs had similar heterogeneous distributions of conformations. However, there were significant changes, dominated by a large increase in the population of unstacked bases from ~22 to 59-68%, depending on their subsite locations, when the oligomers were saturated with g5p. Anisotropy data indicated that 2AP in the bound state was less flexible than in the free oligomer. A control oligomer was labeled with 2AP in the loop of the hairpin and showed no significant change in its base stacking upon g5p binding. A proposed model summarizes the data.
Assuntos
2-Aminopurina/química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Proteínas Virais/química , 2-Aminopurina/metabolismo , Dicroísmo Circular , Colífagos/química , Colífagos/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/virologia , Conformação de Ácido Nucleico , Ligação Proteica , Multimerização Proteica , Espectrometria de Fluorescência , Fatores de Tempo , Proteínas Virais/metabolismoRESUMO
RNA conformational dynamics and the resulting structural heterogeneity play an important role in RNA functions, e.g., recognition. Recognition of HIV-1 TAR RNA has been proposed to occur via a conformational capture mechanism. Here, using ultrafast time-resolved fluorescence spectroscopy, we have probed the complexity of the conformational landscape of HIV-1 TAR RNA and monitored the position-dependent changes in the landscape upon binding of a Tat protein-derived peptide and neomycin B. In the ligand-free state, the TAR RNA samples multiple families of conformations with various degrees of base stacking around the three-nucleotide bulge region. Some subpopulations partially resemble those ligand-bound states, but the coaxially stacked state is below the detection limit. When Tat or neomycin B binds, the bulge region as an ensemble undergoes a conformational transition in a position-dependent manner. Tat and neomycin B induce mutually exclusive changes in the TAR RNA underlying the mechanism of allosteric inhibition at an ensemble level with residue-specific details. Time-resolved anisotropy decay measurements revealed picosecond motions of bases in both ligand-free and ligand-bound states. Mutation of a base pair at the bulge--stem junction has differential effects on the conformational distributions of the bulge bases. A dynamic model of the ensemble view of the conformational landscape for HIV-1 TAR RNA is proposed, and the implication of the general mechanism of RNA recognition and its impact on RNA-based therapeutics are discussed.
Assuntos
Repetição Terminal Longa de HIV , HIV-1/genética , RNA Viral/química , Pareamento de Bases , Sítios de Ligação , HIV-1/química , HIV-1/metabolismo , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Espectrometria de FluorescênciaRESUMO
Leadzyme is a small catalytic RNA that was identified by in vitro selection for Pb2+-dependent cleavage from a tRNA library. Leadzyme employs a unique two-step Pb2+-specific mechanism to cleave within its active site. NMR and crystal structures of the active site revealed different folding patterns, but neither features the in-line alignment for attack by the 2'-OH nucleophilic group. These experimentally determined structures most likely represent ground states and are catalytically inactive. There are significant dynamics of the active site and the motif samples multiple conformations at the ground states. Various metal ion binding sites have been identified, including one that may be occupied by a catalytic Pb2+. Based on functional group analysis, a computational model of the transition state has been proposed. This model features a unique base triple that is consistent with sequence and functional group requirements for catalysis. This structure is likely only populated transiently, but imposing appropriate conformational constraints may significantly stabilize this state thereby promoting catalysis. Other ions may inhibit the cleavage by competing for the Pb2+ binding site, or by stabilizing the ground state thereby suppressing its transition to the catalytically active conformation. Some rare earth ions can enhance the reaction via an unknown mechanism. Because of its unique chemistry and dynamic behavior, leadzyme can continue to serve as an excellent model system for teaching us RNA biology and chemistry.
RESUMO
In bacteriophage λ, interactions between a 22-amino acid peptide (called the N-peptide) and a stem-loop RNA element (called boxB) play a critical role in transcription anti-termination. The N-peptide/boxB complex has been extensively studied, and serves as a paradigm for understanding mechanisms of protein/RNA recognition. Particularly, ultrafast spectroscopy techniques have been applied to monitor picosecond fluorescence decay behaviors of 2-aminopurines embedded at various positions of the boxB RNA. The studies have led to a model in which the bound N-peptide exists in dynamic equilibrium between two states, with peptide C-terminal fragment either stacking on (i.e., the stacked state) or peeling away from (i.e., the unstacked state) the RNA loop. The function of the N-peptide/boxB complex seems to correlate with the fraction of the stacked state. Here, the N-peptide/boxB system is studied using the site-directed spin labeling technique, in which X-band electron paramagnetic resonance spectroscopy is applied to monitor nanosecond rotational behaviors of stable nitroxide radicals covalently attached to different positions of the N-peptide. The data reveal that in the nanosecond regime the C-terminal fragment of bound N-peptide adopts multiple discrete conformations within the complex. The characteristics of these conformations are consistent with the proposed stacked and unstacked states, and their distributions vary upon mutations within the N-peptide. These results suggest that the dynamic two-state model remains valid in the nanosecond regime, and represents a unique mode of function in the N-peptide/boxB RNA complex. It also demonstrates a connection between picosecond and nanosecond dynamics in a biological complex.
Assuntos
Conformação de Ácido Nucleico , Fragmentos de Peptídeos/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Estrutura Secundária de Proteína , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Marcadores de SpinRESUMO
Arginine-rich peptides and small-molecule intercalating agents utilize distinct molecular mechanisms for RNA recognition. Here, we combined these distinct binding modules in an effort to create conjugate ligands with enhanced affinity and specificity using the bacteriophage lambda N peptide-boxB interaction as a model system. We first designed and synthesized a series of peptide-acridine conjugates using portions of the RNA-binding domain of N protein (11- and 22- residue peptide segments) and then compared the binding affinity, specificity, salt dependence, and structural properties of the RNA-peptide and RNA-peptide-acridine conjugate complexes using steady-state fluorescence, CD spectroscopy, NMR, and native gel mobility shift assays (GMSAs). These analyses revealed that the full-length peptide-acridine conjugate displayed substantially improved RNA binding affinity ( approximately 80-fold; K(d) approximately 15 pM) relative to that of the peptide alone (K(d) approximately 1.2 nM). In accordance, we also observed specificity enhancement ( approximately 25-fold) as determined via comparison of the binding of the best conjugate to a cognate lambda boxB RNA with that to a noncognate P22 RNA hairpin (80-fold vs 3.2-fold enhancement). Furthermore, the observed binding enhancement was unique to the full-length conjugate with a flexible linker, implying that the structural context of the acridine presentation was critical. Taken together, our observations support the idea that peptide- and intercalation-based binding can be combined to create a new class of high-affinity, high-specificity RNA-binding ligands.
Assuntos
Acridinas/metabolismo , Bacteriófago lambda/metabolismo , RNA/metabolismo , Bacteriófago lambda/genética , Ligantes , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , RNA/genéticaRESUMO
High-resolution crystal structures and biophysical analyses of purine-sensing riboswitches have revealed that a network of hydrogen bonding interactions appear to be largey responsible for discrimination of cognate ligands against structurally related compounds. Here we report that by using femtosecond time-resolved fluorescence spectroscopy to capture the ultrafast decay dynamics of the 2-aminopurine base as the ligand, we have detected the presence of multiple conformations of the ligand within the binding pockets of one guanine-sensing and two adenine-sensing riboswitches. All three riboswitches have similar conformational distributions of the ligand-bound state. The known crystal structures represent the global minimum that accounts for 50-60% of the population, where there is no significant stacking interaction between the ligand and bases of the binding pocket, but the hydrogen-bonding cage collectively provides an electronic environment that promotes an ultrafast ( approximately 1 ps) charge transfer pathway. The ligand also samples multiple conformations in which it significantly stacks with either the adenine or the uracil bases of the A21-U75 and A52-U22 base pairs that form the ceiling and floor of the binding pocket, respectively, but favors the larger adenine bases. These alternative conformations with well-defined base stacking interactions are approximately 1-1.5 kcal/mol higher in DeltaG degrees than the global minimum and have distinct charge transfer dynamics within the picosecond to nanosecond time regime. Inside the pocket, the purine ligand undergoes dynamic motion on the low nanosecond time scale, sampling the multiple conformations based on time-resolved anisotropy decay dynamics. These results allowed a description of the energy landscape of the bound ligand with intricate details and demonstrated the elastic nature of the ligand recognition mode by the purine-sensing riboswitches, where there is a dynamic balance between hydrogen bonding and base stacking interactions, yielding the high affinity and specificity by the aptamer domain.
Assuntos
2-Aminopurina/química , Adenina/química , Aptâmeros de Nucleotídeos/química , Guanina/química , RNA Catalítico/química , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Polarização de Fluorescência , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Espectrometria de Fluorescência , TermodinâmicaRESUMO
RNAs often exhibit a high degree of conformational dynamics and heterogeneity, leading to a rugged energy landscape. However, the roles of conformational heterogeneity and rapid dynamics in molecular recognition or RNA function have not been extensively elucidated. Ultrafast time-resolved fluorescence spectroscopic experiments were used here to probe picosecond dynamics of the theophylline-binding RNA aptamer. These studies showed that multiple conformations are populated in the free RNA, indicating that this aptamer employs a conformational capture mechanism for ligand binding. The base on residue 27 in an internal loop exists in at least three conformational states in the free RNA, including binding competent and incompetent states that have distinct fluorescence decay signatures indicating different base stacking interactions. Picosecond dynamics were also detected by anisotropy experiments, where these motions indicate additional dynamics for base 27. The picosecond data show that theophylline binding shifts the equilibrium for conformations of base 27 from primarily stacked in the free RNA to mostly unstacked in the RNA-theophylline complex, as observed in the previous NMR structure. In contrast, base 10 in a second internal loop is mostly preorganized in the free RNA, consistent with it being stacked between G11 and G25, as is observed in the bound state. Picosecond dynamics were also measured on a modified aptamer that binds with higher affinity to 3-methylxanthine than theophylline. The modified aptamer shows less heterogeneity in the aptamer-3-methylxanthine complex than what is observed in the theophylline aptamer-theophylline complex.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Teofilina/química , Xantinas/química , Anisotropia , Aptâmeros de Nucleotídeos/química , Cinética , Conformação de Ácido Nucleico , Espectrometria de Fluorescência , Teofilina/metabolismo , Xantinas/metabolismoRESUMO
RNA structures are very dynamic and the dynamic motions result in a heterogeneous conformational ensemble. It is crucial to illustrate the role of conformational dynamics in RNA function. A variety of spectroscopic methods have been used to investigate the dynamic aspects of RNA structures. Recently, ultrafast time-resolved spectroscopy, a well-established technique, has been introduced as a new tool in this field. With femtosecond time-resolution, one can resolve the heterogeneous nature of RNA conformational ensemble quantitatively, detect and characterize minor unusual conformations, and capture folding events that may occur on a fast timescale. Here, we review the technical aspects of using an ultrafast fluorescence up-conversion technique to probe the heterogeneous base stacking patterns in RNA motifs and dynamic base motions that allow sampling of multiple states.
Assuntos
Conformação de Ácido Nucleico , RNA Viral/química , RNA/química , Espectrometria de Fluorescência/métodos , 2-Aminopurina/química , Adenina/análogos & derivados , Adenina/química , Anisotropia , Desenho de Equipamento , Guanina/análogos & derivados , Guanina/química , Modelos Moleculares , Modelos Estatísticos , Conformação Molecular , Estrutura Molecular , Fatores de TempoRESUMO
The dynamic nature of ribozymes represents a significant challenge in elucidating their structure-dynamics-function relationship. Here, using femtosecond time-resolved spectroscopy and other biophysical tools, we demonstrate that the active site of leadzyme does not have a unique structure, but rather samples an ensemble of conformations that undergo picosecond structural changes. Various base modifications have a profound context-dependent impact on the catalysis.
Assuntos
RNA Catalítico/química , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Cristalografia por Raios X , Hidrólise , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , RNA Catalítico/metabolismo , Análise Espectral/métodosRESUMO
RNA conformational dynamics have been a subject of extensive biophysical studies by spectroscopic and computational techniques. Application of femtosecond time-resolved spectroscopy is a new addition to this field, where the technique is specifically designed to probe the ruggedness of RNA conformational landscape and recognition modes. Such technique allows resolution of heterogeneous conformations within a complex ensemble. Recent applications on GNRA tetraloops, dangling ends, and RNA-peptide complexes produced more quantitative and dynamic pictures for these motifs and interactions, including detection of alternative structures that have been overlooked. Integration of the ultrafast dynamics approach with other biophysical techniques promises to be a more powerful approach for providing unique insights for addressing the challenging task of decoding RNA conformational dynamics and the understanding of the mechanisms of molecular recognition of RNA.
Assuntos
Conformação de Ácido Nucleico , RNA/química , Bacteriófago lambda/metabolismo , Modelos Moleculares , RNA/metabolismo , Fatores de Tempo , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Unpaired bases at the end of an RNA duplex (dangling ends) can stabilize the core duplex in a sequence-dependent manner and are important determinants of RNA folding, recognition, and functions. Using 2-aminopurine as a dangling end purine base, we have employed femtosecond time-resolved fluorescence spectroscopy, combined with UV optical melting, to quantitatively investigate the physical and structural nature of the stacking interactions between the dangling end bases and the terminal base pairs. A 3'-dangling purine base has a large subpopulation that stacks on the guanine base of the terminal GC or UG pair, either intrastrand or cross-strand depending on the orientation of the pair, thus providing stabilization of different magnitudes. On the contrary, a 5'-dangling purine base only has a marginal subpopulation that stacks on the purine of the same strand (intrastrand) but has little cross-strand stacking. Thus a 5'-dangling purine does not provide significant stabilization. These stacking structures are not static, and a dangling end base samples a range of stacked and unstacked conformations with respect to the terminal base pair. Femtosecond time-resolved anisotropy decay reveals certain hindered base conformational dynamics that occur on the picosecond to nanosecond time scales, which allow the dangling base to sample these substates. When the dangling purine is opposite to a U and is able to form a potential base pair at the end of the duplex, there is an interplay of base stacking and hydrogen-bonding interactions that depends on the orientation of the base pair relative to the adjacent GC pair. By resolving these populations that are dynamically exchanging on fast time scales, we elucidated the correlation between dynamic conformational distributions and thermodynamic stability.
Assuntos
RNA/química , Anisotropia , Sequência de Bases , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA/metabolismo , Ribonucleotídeos/química , Relação Estrutura-Atividade , TermodinâmicaRESUMO
NMR studies provide insights into structural features of internal loops. These insights can be combined with thermodynamic studies to generate models for predicting structure and energetics. The tandem mismatch internal loop, 5'GUGG3'(3'CUAC5'), has been studied by NMR. The NMR structure reveals an internal loop with no hydrogen bonding between the loop bases and with the G in the AG mismatch flipped out of the helix. The sequence of this internal loop is highly conserved in rRNA. The loop is located in the large ribosomal subunit and is part of a conserved 58-nt fragment that is the binding domain of ribosomal protein L11. Structural comparisons between variants of this internal loop in crystal structures of the 58-nt domain complexed with L11 protein and of the large ribosomal subunit (LSU) suggest that this thermodynamically destabilizing internal loop is partially preorganized for tertiary interactions and for binding L11. A model for predicting the base pairing and free energy of 2 x 2 nucleotide internal loops with a purine-purine mismatch next to a pyrimidine-pyrimidine mismatch is proposed on the basis of the present NMR structure and previously reported thermodynamics.
Assuntos
Pareamento Incorreto de Bases , Ressonância Magnética Nuclear Biomolecular , RNA Ribossômico/química , Subunidades Ribossômicas Maiores/genética , Animais , Sequência de Bases , Sequência Conservada , Giardia lamblia/genética , Ligação de Hidrogênio , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Sulfolobus acidocaldarius/genética , Xenopus laevis/genética , Zea mays/genéticaRESUMO
The transcription antiterminator N protein from bacteriophage lambda uses its arginine-rich motif to specifically bind a stem-loop RNA hairpin (boxB) as a bent alpha-helix. A single stacking interaction between a tryptophan (Trp-18) and an adenosine (A7) in the RNA loop is robust and necessary for antitermination activity in vivo. Previously, femtosecond fluorescence up-conversion experiments from this laboratory indicated that the N/boxB complex exists in a dynamical two-state equilibrium between stacked and unstacked conformations and that the extent of stacking depends on the identity of peptide residues 14 and 15. In the present work, we have combined transient absorption and fluorescence up-conversion to determine the nature of interactions responsible for this sequence-dependent behavior. Analysis of mutant complexes supports the idea that the beta-carbon of residue 14 enforces the stacked geometry by hydrophobic interaction with the ribose of A7, whereas a positive charge at this residue plays only a secondary role. A positive charge at position 15 substantially disfavors the stacked state but retains much of the binding energy. Remarkably, in vivo antitermination experiments show strong correlation with our femtosecond dynamics, demonstrating how conformational interplay can control the activity of a macromolecular machine.
Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Interações Hidrofóbicas e Hidrofílicas , Lasers , Conformação Proteica , Proteínas de Ligação a RNA/química , Análise Espectral , Fatores de Tempo , Transcrição Gênica , Proteínas Virais Reguladoras e Acessórias/químicaRESUMO
Femtosecond to nanosecond dynamics of O(2) rebinding to human WT myoglobin and its mutants, V68F and I107F, have been studied by using transient absorption. The results are compared with NO rebinding. Even though the immediate environment around the heme binding site is changed by the mutations, the picosecond geminate rebinding of oxygen is at most minimally affected. On the other hand, the V68F (E11) mutation causes drastic differences in rebinding on the nanosecond time scale, whereas the effect of the I107F (G8) mutation remains relatively small within our 10-ns time window. Unlike traditional homogeneous kinetics and molecular dynamics collisional simulations, we propose a "bifurcation model" for populations of directed and undirected dynamics on the ultrafast time scale, reflecting the distribution of initial protein conformations. The major mutation effect occurs on the time scale on which global protein conformational change is possible, consistent with transitions between the conformations of directed and undirected population playing a role in the O(2) binding. We discuss the relevance of these findings to the bimolecular function of the protein.