Translational recognition of messenger ribonucleic acid caps as a function of pH

Abstract

The degree to which cell-free translation of eukaryotic mRNA is stimulated by the presence of a 5'-terminal 7-methylguanosine-containing cap is affected by a variety of factors including ionic strength, temperature, mRNA concentration, and the type of mRNA. In this report, we show that pH also affects cap dependence. Translation of globin mRNA from which the cap had been enzymatically removed was relatively insensitive to pH compared with capped mRNA. Also, at low pH (6.6-7.2), the cap analogue m7GTP caused little inhibition of globin mRNA translation in a cell-free system whereas at higher pH the degree of inhibition increased. Finally, the overall extent to which globin mRNA translation could be inhibited at saturating concentrations of m7GTP increased with increasing pH. It is also shown that the pKa of the N-1 proton of m7GTP is affected by mono- and divalent cations. At the K+ and Mg2+ concentrations optimal for cell-free translation, the pKa is approximately 7.4. Since the pH optimum for translation is near 7.6, both keto and enolate forms of m7G are present in appreciable amounts. One interpretation for the observed change in cap dependence with pH is that only the enolate form of m7G is recognized by the cap-binding protein.