Dissociative Transition State in Hepatitis Delta Virus Ribozyme Catalysis.

Ribonucleases and small nucleolytic ribozymes are both able to catalyze RNA strand cleavage through 2'--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 O 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, Zn 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.

Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution.

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 ( = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop.

Analysis of the RNA Binding Specificity Landscape of C5 Protein Reveals Structure and Sequence Preferences that Direct RNase P Specificity.

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.

Determination of the Specificity Landscape for Ribonuclease P Processing of Precursor tRNA 5' Leader Sequences.

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.

Determination of relative rate constants for in vitro RNA processing reactions by internal competition.

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.

Alternative substrate kinetics of Escherichia coli ribonuclease P: determination of relative rate constants by internal competition.

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.

Differential cerebral reactivity to shortest and longer tones: neuromagnetic and behavioral evidence.

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.

Memory-based mismatch response to changes in duration of auditory stimuli: an MEG study.

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).