APPLICATION OF SOLUTION EQUILIBRIUM-ANALYSIS TO IN-VITRO RNA-TRANSCRIPTION

Solution equilibrium analysis of in vitro RNA transcription has been a pplied to examine changes in pH, free magnesium concentration, and con centrations of all chemical ionization species as a transcription reac tion proceeds. With this method, the progress of a transcription react ion can be accurately determined as a function of measured pH. In addi tion, it is demonstrated that this method has significant value as a t ool for achieving improved understanding of the effects of varying sol ution conditions on the dynamics of RNA transcription. Magnesium conce ntration was found to be a critical factor for efficient transcription . Below 5 mM free Mg2+ concentration, the transcription rate and the e fficiency at which nucleoside triphosphates (NTPs) are incorporated ar e 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 prima rily due to sequestering of magnesium. The PPi forms a precipitate wit h magnesium which was; determined to have a molar composition of 2:1 o f Mg:PPi. Transcription rate and efficiency of NTP incorporation are a lso reduced with increasing ionic strength. It is shown that these red uctions can be partially alleviated by replacing chloride with acetate anions.