RESUMO
We describe a chemical coupling procedure that allows joining of two RNAs, one of which contains a site-specific base analog substitution, in the absence of divalent ions. This method allows incorporation of nucleotide analogs at specific positions even into large, cis-cleaving ribozymes. Using this method we have studied the effects of substitution of G638 in the cleavage site loop of the VS ribozyme with a variety of purine analogs having different functional groups and pK(a) values. Cleavage rate versus pH profiles combined with kinetic solvent isotope experiments indicate an important role for G638 in proton transfer during the rate-limiting step of the cis-cleavage reaction.
Assuntos
Endorribonucleases/química , Endorribonucleases/metabolismo , Neurospora/enzimologia , RNA Catalítico/química , RNA Catalítico/metabolismo , RNA Fúngico/química , RNA Fúngico/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Endorribonucleases/genética , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neurospora/genética , Conformação de Ácido Nucleico , Nucleotídeos de Purina/química , RNA Catalítico/genética , RNA Fúngico/genéticaRESUMO
Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two three-way junctions. Second, catalytic activity of the ribozyme is essential for replication of VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that the VS ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS ribozyme favor cleavage of the substrate downstream of the ribozyme core instead. This preference is expected to facilitate processing of the multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.
Assuntos
Endorribonucleases/química , Neurospora , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Fúngico/química , Sequência de Bases , Catálise , Endorribonucleases/genética , Endorribonucleases/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neurospora/enzimologia , Neurospora/genética , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA não Traduzido/química , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
RNA editing by members of the ADAR (adenosine deaminase that acts on RNA) enzyme family involves hydrolytic deamination of adenosine to inosine within the context of a double-stranded pre-mRNA substrate. Editing of the human GluR-B transcript is catalyzed by the enzyme ADAR2 at the Q/R and R/G sites. We have established a minimal RNA substrate for editing based on the R/G site and have characterized the interaction of ADAR2 with this RNA by gel shift, kinetic, and cross-linking analyses. Gel shift analysis revealed that two complexes are formed on the RNA as protein concentration is increased; the ADAR monomers can be cross-linked to one another in an RNA-dependent fashion. We performed a detailed kinetic study of the editing reaction; the data from this study are consistent with a reaction scheme in which formation of an ADAR2.RNA ternary complex is required for efficient RNA editing and in which formation of this complex is rate determining. These observations suggest that RNA adenosine deaminases function as homodimers on their RNA substrates and may partially explain regulation of RNA editing in these systems.
Assuntos
Adenosina Desaminase/metabolismo , Adenosina/metabolismo , Inosina/metabolismo , Edição de RNA/genética , RNA Mensageiro/genética , Receptores de AMPA/metabolismo , Adenosina Desaminase/química , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Humanos , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Pichia/genética , Pichia/metabolismo , Ligação Proteica , Splicing de RNA , RNA Mensageiro/química , Proteínas de Ligação a RNA , Receptores de AMPA/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Most of the small ribozymes, including those that have been investigated as potential therapeutic agents, appear to be rather poor catalysts. These RNAs use an internal phosphoester transfer mechanism to catalyze site-specific RNA cleavage with apparent cleavage rate constants typically <2 min(-1). We have identified variants of one of these, the Neurospora Varkud satellite ribozyme, that self-cleaves with experimentally measured apparent rate constants of up to 10 s(-1) (600 min(-1)), approximately 2 orders of magnitude faster than any previously characterized self-cleaving RNA. We describe structural features of the cleavage site loop and an adjacent helix that affect the apparent rate constants for cleavage and ligation and the equilibrium between them. These data show that the phosphoester transfer ribozymes can catalyze reactions with rate constants much larger than previously appreciated and in the range of those of protein enzymes that perform similar reactions.