Application of solution equilibrium analysis to in vitro RNA transcription

Solution equilibrium analysis of in vitro RNA transcription has been applied to examine changes in pH, free magnesium concentration, and concentrations of all chemical ionization species as a transcription reaction proceeds. With this method, the progress of a transcription reaction can be accuratel...

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Bibliographic Details
Published in:Biotechnology progress 1997-11, Vol.13 (6), p.747-756
Main Authors: KERN, J. A, DAVIS, R. H
Format: Article
Language:English
Subjects:
Base Sequence
Biological and medical sciences
Biotechnology
Chemical Precipitation
Diverse techniques
DNA
Fundamental and applied biological sciences. Psychology
Genetic engineering
Genetic technics
Hydrogen-Ion Concentration
Kinetics
Magnesium - administration & dosage
Magnesium - pharmacology
Methods. Procedures. Technologies
Miscellaneous
Molecular and cellular biology
Nucleotides - metabolism
Osmolar Concentration
Phosphates - metabolism
RNA - genetics
RNA - metabolism
Solutions
Synthetic digonucleotides and genes. Sequencing
Templates, Genetic
Transcription, Genetic
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Summary:Solution equilibrium analysis of in vitro RNA transcription has been applied to examine changes in pH, free magnesium concentration, and concentrations of all chemical ionization species as a transcription reaction proceeds. With this method, the progress of a transcription reaction can be accurately determined as a function of measured pH. In addition, it is demonstrated that this method has significant value as a tool for achieving improved understanding of the effects of varying solution conditions on the dynamics of RNA transcription. Magnesium concentration was found to be a critical factor for efficient transcription. Below 5 mM free Mg2+ concentration, the transcription rate and the efficiency at which nucleoside triphosphates (NTPs) are incorporated are greatly reduced. While inorganic pyrophosphate (PPi), a byproduct of the reaction, was found to directly inhibit the rate of transcription, its detrimental effects on transcription were determined to be primarily due to sequestering of magnesium. The PPi forms a precipitate with magnesium which was determined to have a molar composition of 2:1 of Mg:PPi. Transcription rate and efficiency of NTP incorporation are also reduced with increasing ionic strength. It is shown that these reductions can be partially alleviated by replacing chloride with acetate anions.
ISSN:8756-7938
1520-6033
DOI:10.1021/bp970094p