RESUMO
Heterochromatin affects genome function at many levels. It enables heritable gene repression, maintains chromosome integrity and provides mechanical rigidity to the nucleus1,2. These diverse functions are proposed to arise in part from compaction of the underlying chromatin2. A major type of heterochromatin contains at its core the complex formed between HP1 proteins and chromatin that is methylated on histone H3, lysine 9 (H3K9me). HP1 is proposed to use oligomerization to compact chromatin into phase-separated condensates3-6. Yet, how HP1-mediated phase separation relates to chromatin compaction remains unclear. Here we show that chromatin compaction by the Schizosaccharomyces pombe HP1 protein Swi6 results in phase-separated liquid condensates. Unexpectedly, we find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a nucleosome. Restraining these dynamics impairs compaction of chromatin into liquid droplets by Swi6. Our results indicate that Swi6 couples its oligomerization to the phase separation of chromatin by a counterintuitive mechanism, namely the dynamic exposure of buried nucleosomal regions. We propose that such reshaping of the octamer core by Swi6 increases opportunities for multivalent interactions between nucleosomes, thereby promoting phase separation. This mechanism may more generally drive chromatin organization beyond heterochromatin.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/química , Heterocromatina/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces , Proteínas Cromossômicas não Histona/química , Heterocromatina/genética , Histonas/química , Histonas/metabolismo , Modelos Moleculares , Schizosaccharomyces/química , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Solventes/química , Solventes/metabolismoRESUMO
SWI-SNF alters DNA-histone interactions within a nucleosome in an ATP-dependent manner. These alterations cause changes in the topology of a closed circular nucleosomal array that persist after removal of ATP from the reaction. We demonstrate here that a remodeled closed circular array will revert toward its original topology when ATP is removed, indicating that the remodeled array has a higher energy than that of the starting state. However, reversion occurs with a half-life measured in hours, implying a high energy barrier between the remodeled and standard states. The addition of competitor DNA accelerates reversion of the remodeled array by more than 10-fold, and we interpret this result to mean that binding of human SWI-SNF (hSWI-SNF), even in the absence of ATP hydrolysis, stabilizes the remodeled state. In addition, we also show that SWI-SNF is able to remodel a closed circular array in the absence of topoisomerase I, demonstrating that hSWI-SNF can induce topological changes even when conditions are highly energetically unfavorable. We conclude that the remodeled state is less stable than the standard state but that the remodeled state is kinetically trapped by the high activation energy barrier separating it from the unremodeled conformation.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares , Nucleossomos/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/química , DNA/metabolismo , DNA Helicases , Estabilidade de Medicamentos , Células HeLa , Histonas/metabolismo , Humanos , Técnicas In Vitro , Conformação de Ácido Nucleico , Nucleossomos/química , Plasmídeos/química , Plasmídeos/metabolismoRESUMO
The histone N-terminal tails have been shown previously to be important for chromatin assembly, remodeling, and stability. We have tested the ability of human SWI-SNF (hSWI-SNF) to remodel nucleosomes whose tails have been cleaved through a limited trypsin digestion. We show that hSWI-SNF is able to remodel tailless mononucleosomes and nucleosomal arrays, although hSWI-SNF remodeling of tailless nucleosomes is less effective than remodeling of nucleosomes with tails. Analogous to previous observations with tailed nucleosomal templates, we show both (i) that hSWI-SNF-remodeled trypsinized mononucleosomes and arrays are stable for 30 min in the remodeled conformation after removal of ATP and (ii) that the remodeled tailless mononucleosome can be isolated on a nondenaturing acrylamide gel as a novel species. Thus, nucleosome remodeling by hSWI-SNF can occur via interactions with a tailless nucleosome core.
Assuntos
Cromatina , Proteínas Nucleares/metabolismo , Nucleossomos , Adenosina Trifosfatases/metabolismo , DNA Helicases , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Substâncias Macromoleculares , Moldes Genéticos , Fatores de Transcrição/metabolismo , TripsinaRESUMO
ATP-dependent chromatin remodeling complexes carry out diverse transformations of chromatin. Understanding their mechanisms requires assays that can monitor the kinetics or chromatin remodeling. In this chapter, we describe complimentary native gel-based and FRET-based methods for assaying the kinetics of ATP-driven nucleosome sliding. These methods can be readily adapted to investigate other types of nucleosomal transformations carried out by chromatin remodeling ATPases.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Transferência Ressonante de Energia de Fluorescência/métodos , Nucleossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cinética , XenopusRESUMO
A classic approach in biology, both organismal and cellular, is to compare morphologies in order to glean structural and functional commonalities. The comparative approach has also proven valuable on a molecular level. For example, phylogenetic comparisons of RNA sequences have led to determination of conserved secondary and even tertiary structures, and comparisons of protein structures have led to classifications of families of protein folds. Here we take this approach in a mechanistic direction, comparing protein and RNA enzymes. The aim of comparing RNA and protein enzymes is to learn about fundamental physical and chemical principles of biological catalysis. The more recently discovered RNA enzymes, or ribozymes, provide a distinct perspective on long-standing questions of biological catalysis. The differences described in this review have taught us about the aspects of RNA and proteins that are distinct, whereas the common features have helped us to understand the aspects that are fundamental to biological catalysis. This has allowed the framework that was put forth by Jencks for protein catalysts over 20 years ago (1) to be extended to RNA enzymes, generalized, and strengthened.
Assuntos
Enzimas/metabolismo , RNA Catalítico/metabolismo , Animais , Catálise , HumanosRESUMO
Binding of the Tetrahymena ribozyme's oligonucleotide substrate represents a local folding event in the context of a globally folded RNA. Substrate binding involves P1 duplex formation with the ribozyme's internal guide sequence to give an "open complex', followed by docking of the P1 duplex into tertiary interactions to give a "closed complex'. We have isolated the open complex as a thermodynamically stable species using a site-specific modification and high Na+ concentrations. This has allowed characterization of P1 docking, which represents a folding transition between local secondary and local tertiary structure. P1 docking is entropically driven, possibly accompanied by a release of bound water molecules. Strategies analogous to those described here can be used more generally to study local folding events in large structured RNAs and to explore the structural and energetic landscape for RNA folding.
Assuntos
Conformação de Ácido Nucleico , Oligonucleotídeos/química , RNA Catalítico/química , Animais , Sequência de Bases , Temperatura Baixa , Reagentes de Ligações Cruzadas , Cinética , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Desnaturação de Ácido Nucleico , Oligonucleotídeos/metabolismo , Cloreto de Sódio/farmacologia , Tetrahymena/química , Tetrahymena/enzimologia , TermodinâmicaRESUMO
It has been suggested that the fundamental feature that distinguishes enzymes from simple chemical catalysts is the ability of enzymes to use binding interactions for catalysis. Results with the Tetrahymena group I RNA enzyme described herein directly demonstrate the catalytic contributions of binding interactions. With wild-type ribozyme, specific functional groups at a distance from the site of chemical transformation facilitate substrate binding without accelerating reaction of bound substrate; with modified ribozymes, these functional groups provide the same overall energetic effect but instead accelerate reaction of bound substrate without increasing binding. These observations are quantitatively described by a structural framework that was established by previous results. The P1 duplex between the substrate and the ribozyme's recognition sequence exists in two states, the open complex, in which the substrate is localized to the ribozyme solely by base-pairing interactions, or the closed complex, in which the duplex is docked into tertiary interactions and positioned with respect to the catalytic groups in the active site. In the absence of sufficient binding energy to ensure stable docking in the ground state, added P1 functional groups accelerate reaction of the bound substrate by helping to overcome the energetic barrier for docking into the reactive, closed complex. When the functional groups present on the P1 duplex are sufficient to ensure stable docking in the closed complex, added functional groups give stronger binding without accelerating reaction of the bound substrate. This behavior is a manifestation of the inextricable link between binding interactions and catalysis. The conclusions also have implications for interpreting the effects of site-directed mutagenesis and for the evolution of active site interactions.
Assuntos
RNA Catalítico/química , RNA Catalítico/metabolismo , Animais , Sítios de Ligação , Catálise , Transferência de Energia , Modelos Químicos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , RNA de Protozoário/química , Relação Estrutura-Atividade , Especificidade por Substrato , Tetrahymena/enzimologiaRESUMO
Phylogenetic conservation among > 100 group I introns and previous in vitro studies have implicated a G.U pair as defining the 5'-splice site for exon ligation. The U residue defines the 3' end of the 5' exon, and the complementary G residue is part of the internal guide sequence (IGS) that base pairs to the 5' exon. We now quantitate the effect of this pair on individual reaction steps using the L-21ScaI ribozyme, which is derived from the group I intron of Tetrahymena thermophila pre-rRNA. The following results indicate that interactions with this G.U pair contribute to the binding of the 5'-exon, the positioning of the 5'-splice site with respect to the catalytic site, and the chemical step. The oligonucleotide, CCCUCU, binds to the ribozyme approximately 20-fold stronger than CCCUCC despite the fact that the U-containing oligonucleotide forms an approximately 5-fold less stable duplex with an oligonucleotide analog of the IGS, GGAGGG. This and two independent experimental observations indicate that the G.U pair contributes approximately 100-fold (3 kcal/mol, 50 degrees C) to tertiary interactions that allow the P1 duplex, which is formed between the 5'-exon and the IGS, to dock into the ribozyme's core. The approximately 50-80-fold increase in miscleavage of 5'-exon analogs upon replacement of the 3'-terminal U of CCCUCU with C or upon removal of the 3'-terminal U suggests that the tertiary interactions with the G.U pair not only contribute to docking but also ensure correct positioning of the 5'-splice site with respect to the catalytic site, thereby minimizing the selection of incorrect splice sites. Comparison of the rates of the chemical cleavage step with G.U vs G.C suggests that the G.U pair contributes approximately 10-fold to the chemical step. It was previously suggested that the 2'-hydroxyl of this U residue helps stabilize the 3'-oxyanion leaving group in the chemical transition state via an intramolecular hydrogen bond. Relative reactivities of oligonucleotide substrates with ribose and deoxyribose U and C are consistent with a model based on a recent X-ray crystallographic structure in which the exocyclic amino group of G helps orient the 2'-hydroxyl of U via a bridging water molecule, thereby strengthening the hydrogen bond donated from the 2'-hydroxyl group to the neighboring incipient 3'-oxyanion. Finally, kinetic and thermodynamic evidence for the formation of a G.C+ wobble pair is presented.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Guanina/metabolismo , Splicing de RNA , RNA de Protozoário/metabolismo , Uridina/metabolismo , Animais , Composição de Bases , Sequência de Bases , Desoxirribose/metabolismo , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Íntrons , Modelos Químicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Catalítico/metabolismo , Tetrahymena thermophilaRESUMO
ATP-dependent nucleosome remodeling complexes can be grouped into several classes that may differ in their biochemical remodeling activities and biological roles. Although there are a number of biochemical studies of each class of remodeler, there are very little data directly comparing the biochemical activities of remodelers from different classes. We have purified two ATP-hydrolyzing proteins, SNF2H and BRG1, which are members of complexes from two different classes of remodelers. Consistent with previous reports, these two homogeneous proteins can perform remodeling functions. We show significant functional differences between SNF2H and BRG1 in vitro; although both SNF2H and BRG1 hydrolyze ATP and remodel linear arrays of nucleosomes, only BRG1 can remodel mononucleosomes. Also, only BRG1 can alter the topology of nucleosomal plasmids. We propose that these functional differences reflect significant mechanistic differences between the two remodeler classes that will impact their biological roles.
Assuntos
Adenosina Trifosfatases/química , Proteínas Cromossômicas não Histona/química , Proteínas Nucleares/química , Fatores de Transcrição/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Clonagem Molecular , DNA/metabolismo , DNA Helicases , Relação Dose-Resposta a Droga , Glicerol/metabolismo , Humanos , Hidrólise , Insetos , Cinética , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , Ligação Proteica , Fatores de TempoRESUMO
We have dissected the steps in nucleosome remodeling by BRG1, the ATPase subunit of human SWI/SNF. BRG1-catalyzed DNA exposure is not enhanced by the proximity of the site to the ends of nucleosomal DNA, suggesting that the mechanism involves more than peeling or sliding of the DNA. Comparison of DNA exposure at specific sites with overall changes in the path of DNA implies that BRG1 generates multiple distinct remodeled structures and continuously interconverts them. These characteristics are shared by the entire SWI/SNF complex and have parallels, as well as interesting differences, with the activities of GroEL and Hsp70 protein chaperones. The chaperone-like activity of SWI/SNF is expected to create multiple opportunities for the binding of distinct regulatory factors, providing one mechanism by which SWI/SNF family complexes can contribute to both activation and repression of transcription.
Assuntos
Cromatina/química , Cromatina/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Catálise , Linhagem Celular , Cromatina/genética , DNA/química , DNA/genética , DNA Helicases , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Hidrólise , Cinética , Substâncias Macromoleculares , Modelos Genéticos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Conformação Molecular , Ensaios de Proteção de Nucleases , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Subunidades Proteicas , Termodinâmica , Fatores de Transcrição/químicaRESUMO
Binding of the Tetrahymena ribozyme's oligonucleotide substrate (S) involves P1 duplex formation with the ribozyme's internal guide sequence (IGS) to give an open complex, followed by docking of the P1 duplex into the catalytic core via tertiary interactions to give a closed complex. The overall binding energies provided by 2' OH groups on S and IGS have been measured previously. To obtain the energetic contribution of each of these 2' OH groups in the docking step, we have separately measured their contribution to the stability of a model P1 duplex using "substrate inhibition". This new approach allows measurement of duplex stabilities under conditions identical to those used for ribozyme binding measurements. The tertiary binding energies from the individual 2' OH groups include a small destabilizing contribution of 0.7 kcal/mol and stabilizing contributions of up to -2.9 kcal/mol. The energetic contributions of specific 2' OH groups are discussed in the context of considerable previous work that has characterized the tertiary interactions of the P1 duplex. A "threshold" model for the open and closed complexes is presented that provides a framework to interpret the energetic effects of functional group substitutions on the P1 duplex. The sum of the tertiary stabilization provided by the conserved G x U wobble at the cleavage site and the individual 2' OH groups on the P1 duplex is significantly greater than the observed tertiary stabilization of S (11.0 vs 2.2 kcal/mol). It is suggested that there is an energetic cost for docking the P1 duplex into the active site that is paid for by the "intrinsic binding energy" of groups on the P1 duplex. Substrates that lack sufficient tertiary binding energy to overcome this energetic barrier exhibit reduced reactivities. Thus, the ribozyme appears to use the intrinsic binding energy of groups on the P1 duplex for catalysis. This intrinsic binding energy may be used to position reactants within the active site and to induce electrostatic destabilization of the substrate, relative to its interactions in solution.
Assuntos
Transferência de Energia , Radical Hidroxila/química , Conformação de Ácido Nucleico , RNA Catalítico/química , Tetrahymena/genética , Animais , Ligação Competitiva , Catálise , Modelos Químicos , Oligonucleotídeos/antagonistas & inibidores , Oligonucleotídeos/química , RNA Catalítico/antagonistas & inibidores , Ribonuclease T1/química , Especificidade por SubstratoRESUMO
The Tetrahymena group I ribozyme's oligonucleotide substrate, CCCUCUA(5), forms six base pairs with the ribozyme's internal guide sequence (IGS, 5'GGAGGG) to give the P1 duplex, and this duplex then docks into the active site via tertiary interactions. Shortening the substrate by three residues to give UCUA(5) reduces the equilibrium constant for P1 docking by approximately 200-fold even though UCUA(5) retains all the functional groups known to be involved in tertiary interactions [Narlikar, G. J., Bartley, L. E., Khosla, M., and Herschlag, D. (1999) Biochemistry 38, 14192-14204]. Here we show that the P1 duplex formed with UCUA(5) engages in all of the major tertiary interactions made by the standard P1 duplex. This suggests that the destabilization is not due to disruption of specific tertiary interactions. It therefore appears that the weaker docking of UCUA(5) arises from the increased conformational freedom of the undocked P1 duplex, which has three unpaired IGS residues and thus a larger entropic cost for docking. Further, a 2'-methoxy substitution at an IGS residue that is base-paired in the standard P1 duplex with CCCUCUA(5) but unpaired in the P1 duplex with UCUA(5) destabilizes docking of the standard P1 duplex approximately 300-fold more than it destabilizes docking of the P1 duplex formed with UCUA(5). These results suggest that fixation of groups in the context of a rigid duplex may be a general strategy used by RNA to substantially increase interaction specificity, both by aiding binding of the desired functional groups and by increasing the energetic cost of forming alternative interactions.
Assuntos
Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , RNA Catalítico/química , Animais , Sítios de Ligação , Cinética , Ácidos Nucleicos Heteroduplexes/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , RNA Catalítico/metabolismo , Especificidade por Substrato , TetrahymenaRESUMO
We have probed the electrostatic environment of the active site of the Tetrahymena group I ribozyme (E) using protonated 2'-aminoguanosine (), in which the 2'-OH of the guanosine nucleophile (G) is replaced by an group. At low concentrations of divalent metal ion (2 mM Mg(2+)), binds at least 200-fold stronger than G or G(NH)()2, with a dissociation constant of =1 microM from the ribozyme. oligonucleotide substrate. complex (). This strong binding suggests that the group interacts with negatively charged phosphoryl groups within the active site. Increasing the concentration of divalent metal ion weakens the binding of to E. S more than 10(2)-fold. The Mn(2+) concentration dependence suggests that M(C), the metal ion that interacts with the 2'-moiety of G in the normal reaction, is responsible for this effect. M(C) and compete for binding to the active site; this competition could arise from electrostatic repulsion between the positively charged and M(C) and, possibly, from their competition for interaction with active site phosphoryl groups. The reactive phosphoryl group of S increases the competition between M(C) and, consistent with a network of interactions involving M(C) that help position the reactive phosphoryl group and the guanosine nucleophile with respect to one another. The chemical step with bound is at least 10(4)-fold slower than with G or G(NH)()2. These results provide additional support for an integral role of M(C) in catalysis by the Tetrahymena ribozyme, and demonstrate the utility of the moiety as an electrostatic probe within a structured RNA.
Assuntos
Guanosina/metabolismo , Prótons , RNA Catalítico/metabolismo , Tetrahymena/enzimologia , Aminas , Animais , Sítios de Ligação , Guanosina/análogos & derivados , Guanosina/química , Magnésio/química , Magnésio/metabolismo , Manganês/química , Manganês/metabolismo , Modelos Químicos , Oligorribonucleotídeos/metabolismo , RNA Catalítico/química , Soluções , Eletricidade Estática , Especificidade por SubstratoRESUMO
A fundamental catalytic principle for protein enzymes in the use of binding interactions away from the site of chemical transformation for catalysis. We have compared the binding and reactivity of a series of oligonucleotide substrates and products of the Tetrahymena ribozyme, which catalyzes a site-specific phosphodiester cleavage reaction: CCCUCUpA+G<-->CCCUCU-OH+GpA. The results suggest that this RNA enzyme, like protein enzymes, can utilize binding interactions to achieve substantial catalysis via entropic fixation and substrate destabilization. The stronger binding of the all-ribose oligonucleotide product compared to an analog with a terminal 3' deoxyribose residue gives an effective concentration of 2200 M for the 3' hydroxyl group, a value approaching those obtained with protein enzymes and suggesting the presence of a structurally well defined active site capable of precise positioning. The stabilization from tertiary binding interactions is 40-fold less for the oligonucleotide substrate than the oligonucleotide product, despite the presence of the reactive phosphoryl group in the substrate. This destabilization is accounted for by a model in which tertiary interactions away from the site of bond cleavage position the electron-deficient 3' bridging phosphoryl oxygen of the oligonucleotide substrate next to an electropositive Mg ion. As the phosphodiester bond breaks and this 3' oxygen atom develops a negative charge in the transition state, the weak interaction of the substrate with Mg2+ becomes strong. These strategies of "substrate destabilization" and "transition state stabilization" provide estimated rate enhancements of approximately 280- and approximately 60-fold, respectively. Analogous substrate destabilization by a metal ion or hydrogen bond donor may be used more generally by RNA and protein enzymes catalyzing reactions of phosphate esters.
Assuntos
RNA Catalítico/química , RNA Catalítico/metabolismo , Tetrahymena/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Catálise , Cinética , Magnésio/metabolismo , Oligorribonucleotídeos/síntese química , RNA Catalítico/biossíntese , Especificidade por Substrato , Transcrição GênicaRESUMO
Protein complexes of the SWI/SNF family remodel nucleosome structure in an ATP-dependent manner. Each complex contains between 8 and 15 subunits, several of which are highly conserved between yeast, Drosophila, and humans. We have reconstituted an ATP-dependent chromatin remodeling complex using a subset of conserved subunits. Unexpectedly, both BRG1 and hBRM, the ATPase subunits of human SWI/SNF complexes, are capable of remodeling mono-nucleosomes and nucleosomal arrays as purified proteins. The addition of INI1, BAF155, and BAF170 to BRG1 increases remodeling activity to a level comparable to that of the whole hSWI/SNF complex. These data define the functional core of the hSWI/SNF complex.
Assuntos
Actinas/química , Cromatina/ultraestrutura , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA/química , Proteínas de Drosophila , Proteínas Nucleares/química , Nucleossomos/ultraestrutura , Proteínas de Ligação a RNA , Ribonucleoproteína Nuclear Pequena U1/química , Fatores de Transcrição/química , Actinas/fisiologia , Trifosfato de Adenosina/fisiologia , Sistema Livre de Células , Cromatina/metabolismo , DNA Helicases , Proteínas de Ligação a DNA/fisiologia , Evolução Molecular , Humanos , Substâncias Macromoleculares , Proteínas Nucleares/fisiologia , Nucleossomos/metabolismo , Proteínas Recombinantes de Fusão/química , Ribonucleoproteína Nuclear Pequena U1/fisiologia , Fatores de Transcrição/fisiologiaRESUMO
Binding of the Tetrahymena group I ribozyme's oligonucleotide substrate occurs in two steps: P1 duplex formation with the ribozyme's internal guide sequence which forms an "open complex" is followed by docking of the P1 duplex into tertiary interactions within the catalytic core which forms a "closed complex". By systematically varying substrate length, pH, and temperature, we have identified conditions under which P1 duplex formation, P1 docking, or the chemical cleavage step limits the rate of the ribozyme reaction. This has enabled characterization of the individual steps as a function of substrate length, pH, and temperature, leading to several conclusions. (1) The rate constant for formation of the open complex is largely independent of substrate length, pH, and temperature, analogous to that of duplex formation in solution. This extends previous results suggesting that open complex formation entails mainly secondary structure formation and strengthens the view that the second binding step, P1 docking, represents a simple transition from secondary to tertiary structure in the context of an otherwise folded RNA. (2) The temperature dependence of the rate constant for P1 docking yields a negative activation entropy, in contrast to the positive entropy change previously observed for the docking equilibrium. This suggests a model in which tertiary interactions are not substantially formed in the transition state for P1 docking. (3) Shortening the substrate by three residues decreases the equilibrium constant for P1 docking by 200-fold, suggesting that the rigidity imposed by full-length duplex formation facilitates formation of tertiary interactions. (4) Once docked, shortened substrates are cleaved at rates within 3-fold of that for the full-length substrate. Thus, all the active site interactions required to accelerate the chemical cleavage event are maintained with shorter substrates. (5) The rate constant of approximately 10(3) min(-1) obtained for P1 docking is significantly faster than the other steps previously identified in the tertiary folding of this RNA. Nevertheless, P1 docking presumably follows other tertiary folding steps because the P1 duplex docks into a core that is formed only upon folding of the rest of the ribozyme.
Assuntos
Conformação de Ácido Nucleico , Oligonucleotídeos/metabolismo , RNA Catalítico/química , RNA Catalítico/metabolismo , RNA de Protozoário/química , RNA de Protozoário/metabolismo , Tetrahymena/enzimologia , Animais , Sítios de Ligação , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Ácidos Nucleicos Heteroduplexes/química , Oligonucleotídeos/química , Relação Estrutura-Atividade , Especificidade por Substrato , Temperatura , Tetrahymena/genética , TermodinâmicaRESUMO
Eukaryotic Rvb1p and Rvb2p are two highly conserved proteins related to the helicase subset of the AAA+ family of ATPases. Conditional mutants in both genes show rapid changes in the transcription of over 5% of yeast genes, with a similar number of genes being repressed and activated. Both Rvb1p and Rvb2p are required for maintaining the induced state of many inducible promoters. ATP binding and hydrolysis by Rvb1p and Rvb2p is individually essential in vivo, and the two proteins are associated with each other in a high molecular weight complex that shows ATP-dependent chromatin remodeling activity in vitro. Our findings show that Rvb1p and Rvb2p are essential components of a chromatin remodeling complex and determine genes regulated by the complex.