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 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.