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

ABSTRACT: 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.

SUBMITTER: Lin HC

PROVIDER: S-EPMC4252809 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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

Lin Hsuan-Chun HC Yandek Lindsay E LE Gjermeni Ino I Harris Michael E ME

Analytical biochemistry 20140828

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 r ...[more]

PMID: 25173512

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Project description:The forward and reverse H + CO ↔ HCO reactions are important for combustion chemistry and have been studied from a wide variety of theoretical and experimental techniques. However, most of the chemical kinetic investigations concerning these processes are focused on low pressures or fall-off regions. Hence, a high-level electronic structure treatment was employed here in order to provide accurate rate constant values for these reactions at the high-pressure limit along temperatures from 50 to 4000 K. In relative terms, the variational effects on rate constants are shown to be almost as important at high temperatures as quantum tunneling corrections at the lowest temperatures investigated. The activation energies fitted by using modified and traditional Arrhenius' equations for the forward rate constants from 298 to 2000 K are, respectively, equal to 2.64 and 3.89 kcal mol-1, while similar fittings provided, respectively, 1.96 and 3.22 kcal mol-1, considering now forward rate constants from a temperature range of 298-373 K. This last activation energy determination (3.22 kcal mol-1) is in better agreement with the commonly referenced experimental value of 2.0 ± 0.4 kcal mol-1, also obtained from traditional fittings in the range 298-373 K, than the value attained from a broader temperature range fitting (3.89 kcal mol-1). However, some additional care must be considered along these comparisons once the experimental reaction rate measurements have been done for the trimolecular H + CO + M → HCO + M reaction instead. Anyway, the usage of appropriate temperature ranges is fundamental for reliable activation energy comparisons, although the remaining deviation between theory and experiment is still large and is possibly caused by the different pressure regimes assessed in each case. Finally, we roughly estimated that the high-pressure limit for the HCO decomposition into H and CO can be achieved along temperatures ranging from ∼246 and ∼255 K downward, respectively, at pressures of 1.1 and 9.6 atm, although further experiments should be carried out in order to improve these estimates. On the other hand, pressures larger than 9.8 × 104 atm are required for the aforementioned dissociation reaction to attain the high-pressure limit at 700 K. Therefore, the rate constants determined here are probably applicable in combustion studies at lower temperatures.

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

Publications

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

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