The Effect of Temperature and Ionic Strength on the Electrophoretic Mobility of Yeast Mitochondrial RNA

TLDR: The results show that judicious variation of temperature and ionic strength may allow the separation on gels of RNA mixtures that cannot be resolved under standard conditions.

Abstract: The electrophoretic mobilities of mitochondrial and cell-sap ribosomal RNAs from Saccharomyces carlsbergensis have been measured relative to the mobility of Escherichia coli rRNA in 2.4% polyacrylamide gels at various temperatures and ionic strengths. At 5° C and in 20 mM sodium acetate, 40 mM Tris-acetate, 2 mM EDTA (pH 7.8), mitochondrial and cell-sap rRNAs co-migrated and were only partially resolved from E. coli rRNA. When the temperature of electrophoresis was increased the mobility of the mitochondrial rRNAs (and to a lesser extent of the cell-sap rRNAs) decreased relative to the mobility of the E. coli rRNA species, reaching a minimum between 9 and 16° C. As a consequence the mitochondrial and cell-sap rRNAs were completely resolved in this buffer at temperatures above 9° C. Similar effects of temperature on relative electrophoretic mobility were observed in 90 mM Tris-borate buffer at pH 8.3 and the effect was even more pronounced at low ionic strength (10 mM NaCl, 5 mM Tris-HCl, 2 mM EDTA, pH 7.5). At low ionic strength the decrease in the relative electrophoretic mobility of mitochondrial rRNA with an increase in temperature was accompanied by a decrease in sedimentation coefficient from 21–22 S (large component) and 14 S (small component) at 5° C to 16 and 11.5 S at 20° C (calculated in relation to assumed S-values of 23 and 16 S for the rRNA species of E. coli). We conclude that the electrophoretic mobility of RNA in polyacrylamide gels is more dependent on secondary structure than previous work had suggested and that caution is needed to interpret the relative mobility of mitochondrial RNA on gels in terms of molecular weight. In addition, our results show that judicious variation of temperature and ionic strength may allow the separation on gels of RNA mixtures that cannot be resolved under standard conditions.