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Application of Solution Equilibrium Analysis to in Vitro RNA Transcription
Author(s) -
Kern Jeffrey A.,
Davis Robert H.
Publication year - 1997
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp970094p
Subject(s) - transcription (linguistics) , chemistry , magnesium , nucleoside , rna , nucleotide , in vitro , ionic strength , pyrophosphate , biochemistry , enzyme , organic chemistry , aqueous solution , gene , philosophy , linguistics
Abstract 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 Mg 2+ concentration, the transcription rate and the efficiency at which nucleoside triphosphates (NTPs) are incorporated are greatly reduced. While inorganic pyrophosphate (PP i ), 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 PP i forms a precipitate with magnesium which was determined to have a molar composition of 2:1 of Mg:PP i . 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.