Application of solution equilibrium analysis to in vitro RNA transcription
Application of solution equilibrium analysis to in vitro RNA transcription
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DOI：
10.1021/bp970094p
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发表时间：
1997-11-01
影响因子：
2.9
通讯作者：
Davis, RH
中科院分区：
文献类型：
--
作者：
Kern, JA;Davis, RH
体外RNA转录的溶液平衡分析已被用于检测转录反应进行时pH、游离镁浓度和所有化学电离物质浓度的变化。通过这种方法，可以准确地确定转录反应的进展，作为测量ph的函数。此外，研究表明，这种方法作为一种工具具有重要的价值，可以更好地理解不同溶液条件对RNA转录动力学的影响。镁浓度是影响转录效率的关键因素。当游离Mg2+浓度低于5 mM时，转录速率和三磷酸核苷（NTPs）的结合效率大大降低。虽然无机焦磷酸盐（PPi）是该反应的副产物，被发现直接抑制转录速率，但其对转录的有害影响被确定主要是由于镁的隔离。PPi与镁形成沉淀；Mg:PPi的摩尔组成为2:1。随着离子强度的增加，NTP的转录速率和结合效率也随之降低。结果表明，用醋酸阴离子代替氯离子可以部分减轻这些减少。
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.