RESUMEN
Ribonucleases and small nucleolytic ribozymes are both able to catalyze RNA strand cleavage through 2'-O-transphosphorylation, provoking the question of whether protein and RNA enzymes facilitate mechanisms that pass through the same or distinct transition states. Here, we report the primary and secondary 18O kinetic isotope effects for hepatitis delta virus ribozyme catalysis that reveal a dissociative, metaphosphate-like transition state in stark contrast to the late, associative transition states observed for reactions catalyzed by specific base, Zn2+ ions, or ribonuclease A. This new information provides evidence for a discrete ribozyme active site design that modulates the RNA cleavage pathway to pass through an altered transition state.
Asunto(s)
ARN Catalítico , ARN Catalítico/química , Virus de la Hepatitis Delta/genética , Virus de la Hepatitis Delta/metabolismo , ARN/química , Catálisis , Dominio Catalítico , Conformación de Ácido Nucleico , CinéticaRESUMEN
Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5'-YAG-3' and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo.
Asunto(s)
Empalme Alternativo , Ribonucleoproteína Nuclear Heterogénea A1/química , Dominios y Motivos de Interacción de Proteínas , ARN Viral/química , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Exones , VIH/genética , VIH/metabolismo , Ribonucleoproteína Nuclear Heterogénea A1/genética , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Humanos , Intrones , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , ARN Viral/genética , ARN Viral/metabolismo , TermodinámicaRESUMEN
A single enzyme, ribonuclease P (RNase P), processes the 5' ends of tRNA precursors (ptRNA) in cells and organelles that carry out tRNA biosynthesis. This substrate population includes over 80 different competing ptRNAs in Escherichia coli. Although the reaction kinetics and molecular recognition of a few individual model substrates of bacterial RNase P have been well described, the competitive substrate kinetics of the enzyme are comparatively unexplored. To understand the factors that determine how different ptRNA substrates compete for processing by E. coli RNase P, we compared the steady state reaction kinetics of two ptRNAs that differ at sequences that are contacted by the enzyme. For both ptRNAs, substrate cleavage is fast relative to dissociation. As a consequence, V/K, the rate constant for the reaction at limiting substrate concentrations, reflects the substrate association step for both ptRNAs. Reactions containing two or more ptRNAs follow simple competitive alternative substrate kinetics in which the relative rates of processing are determined by ptRNA concentration and their V/K. The relative V/K values for eight different ptRNAs, which were selected to represent the range of structure variation at sites contacted by RNase P, were determined by internal competition in reactions in which all eight substrates were present simultaneously. The results reveal a relatively narrow range of V/K values, suggesting that rates of ptRNA processing by RNase P are tuned for uniform specificity and consequently optimal coupling to precursor biosynthesis.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Ribonucleasa P/química , Algoritmos , Secuencia de Bases , Unión Competitiva , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , División del ARN , Procesamiento Postranscripcional del ARN , ARN Bacteriano/química , ARN de Transferencia/químicaRESUMEN
Studies of RNA recognition and catalysis typically involve measurement of rate constants for reactions of individual RNA sequence variants by fitting changes in substrate or product concentration to exponential or linear functions. A complementary approach is determination of relative rate constants by internal competition, which involves quantifying the time-dependent changes in substrate or product ratios in reactions containing multiple substrates. Here, we review approaches for determining relative rate constants by analysis of both substrate and product ratios and illustrate their application using the in vitro processing of precursor transfer RNA (tRNA) by ribonuclease P as a model system. The presence of inactive substrate populations is a common complicating factor in analysis of reactions involving RNA substrates, and approaches for quantitative correction of observed rate constants for these effects are illustrated. These results, together with recent applications in the literature, indicate that internal competition offers an alternate method for analyzing RNA processing kinetics using standard molecular biology methods that directly quantifies substrate specificity and may be extended to a range of applications.
Asunto(s)
Escherichia coli/metabolismo , Precursores del ARN/química , Procesamiento Postranscripcional del ARN , ARN Bacteriano/química , ARN de Transferencia/química , Ribonucleasa P/metabolismo , Emparejamiento Base , Secuencia de Bases , Catálisis , Escherichia coli/genética , Técnicas In Vitro , Datos de Secuencia Molecular , Precursores del ARN/metabolismo , ARN Bacteriano/metabolismo , ARN de Transferencia/metabolismo , Especificidad por Sustrato , TermodinámicaRESUMEN
Maturation of tRNA depends on a single endonuclease, ribonuclease P (RNase P), to remove highly variable 5' leader sequences from precursor tRNA transcripts. Here, we use high-throughput enzymology to report multiple-turnover and single-turnover kinetics for Escherichia coli RNase P processing of all possible 5' leader sequences, including nucleotides contacting both the RNA and protein subunits of RNase P. The results reveal that the identity of N(-2) and N(-3) relative to the cleavage site at N(1) primarily control alternative substrate selection and act at the level of association not the cleavage step. As a consequence, the specificity for N(-1), which contacts the active site and contributes to catalysis, is suppressed. This study demonstrates high-throughput RNA enzymology as a means to globally determine RNA specificity landscapes and reveals the mechanism of substrate discrimination by a widespread and essential RNA-processing enzyme.
Asunto(s)
Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , Ribonucleasa P/metabolismo , Cinética , Especificidad por SustratoRESUMEN
RNA binding proteins (RBPs) are typically involved in non-equilibrium cellular processes, and specificity can arise from differences in ground state, transition state, or product states of the binding reactions for alternative RNAs. Here, we use high-throughput methods to measure and analyze the RNA association kinetics and equilibrium binding affinity for all possible sequence combinations in the precursor tRNA binding site of C5, the essential protein subunit of Escherichia coli RNase P. The results show that the RNA sequence specificity of C5 arises due to favorable RNA-protein interactions that stabilize the transition state for association and bound enzyme-substrate complex. Specificity is further impacted by unfavorable RNA structure involving the C5 binding site in the ground state. The results illustrate a comprehensive quantitative approach for analysis of RNA binding specificity, and show how both RNA structure and sequence preferences of an essential protein subunit direct the specificity of a ribonucleoprotein enzyme.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Precursores del ARN/metabolismo , ARN Bacteriano/metabolismo , Ribonucleasa P/metabolismo , Secuencia de Bases , Sitios de Unión , Escherichia coli/química , Proteínas de Escherichia coli/química , Modelos Moleculares , Conformación Proteica , Precursores del ARN/química , ARN Bacteriano/química , Ribonucleasa P/químicaRESUMEN
Detecting a change in sound duration is important in language processing. The cerebral reactivity to a duration deviant in oddball paradigm has been reflected as a mismatch negativity (MMN). This study aimed to see cerebral responses to several duration-varying sounds presented with equal probability. Magnetoencephalographic (MEG) and behavior responses to equi-probable sounds (25-50-75-100-125 ms or 50-75-100-125-150 ms tones) were recorded in 10 healthy adult volunteers. By subtracting the average of the responses to 4 longer tones from the response to the shortest tone, a clear deflection peaking at 100-200 ms from stimulus onset was identified. This activity was called as sub-standard MMNm, and its amplitude tended to increase with the increment of duration deviance within a stimulation paradigm. The source of sub-standard MMNm was localized in superior temporal area, with 5-6 mm more anterior to the generator of N100m response. Behavioral tests also showed best performance in the recognition of the shortest tone than longer tones. In conclusion, the preferential response to the shortest tone in an equiprobable paradigm suggests an asymmetrical processing in the auditory cortex for duration-varying sounds.
Asunto(s)
Corteza Auditiva/fisiología , Vías Auditivas/fisiología , Magnetoencefalografía , Percepción de la Altura Tonal , Psicoacústica , Detección de Señal Psicológica , Estimulación Acústica , Adulto , Audiometría , Umbral Auditivo , Potenciales Evocados Auditivos , Femenino , Humanos , Masculino , Tiempo de Reacción , Factores de Tiempo , Adulto JovenRESUMEN
OBJECTIVE: Differences in physical features and occurrence probability between standards and deviants in oddball paradigms provide contributions to magnetic mismatch negativity (MMNm). We aimed to reduce these influential factors and extract memory-based MMNm by adding a control paradigm. METHODS: Magnetoencephalographic responses were recorded in 13 healthy adults with an oddball paradigm (125-ms standard and 50-ms deviant tones) and an equiprobable control paradigm (50-ms control and four other duration-varying tones). The stimulus onset asynchrony was 500 ms. Controlled MMNm was obtained by subtracting control-evoked responses from deviant-evoked responses. RESULTS: With respect to the onset of stimulus difference, the peak latency of controlled MMNm was compatible with previous intracranial MMN recordings. Both controlled and traditional MMNm were generated around the superior temporal cortex, whereas the controlled MMNm amplitude was about 70% of traditional MMNm amplitude. Right-hemispheric dominance was observed in traditional MMNm but not in controlled MMNm. N100m amplitude was smaller in standard-evoked than in deviant- or control-evoked responses. CONCLUSIONS: Controlled MMNm reflects memory-based processing of duration changes, whereas traditional MMNm additionally involves non-memory activations related to differential refractoriness states and physical properties between standard and deviant stimuli. SIGNIFICANCE: The memory-based processing of auditory deviants may be preferentially extracted by adding a control paradigm.