Effect of ribonuclease T1 on ribosomal subunits of rat liver

Abstract

The accessibility of 28S RNA within the ribosomal subunits to ribonuclease T1 was studied, in comparing results obtained after enzyme treatment of compact, K+ deficient 60S subunits and of EDTA-treated 60S subunits. RNA, extracted from the subunits, using a mixture of sodium dodecyl sulfate and phenol was analyzed on sucrose gradients. The RNA from active subunits was only degraded in high enzyme concentrations. In the K+ deficient subunits, RNA is more accessible since it breaks down into 6 well-defined fragments, sedimenting between 4S and 18.5S. Within the EDTA-subunits, there is no more protection of the RNA. In fact, it is degraded by weak enzyme concentrations, as is the free 28S RNA, giving heterogeneous fragments. Comparison of the melting curves of subunits and free 28S RNA showed that it is only in EDTA subunits that proteins do not stabilize the secondary structure of RNA. In the case of 40S subunits, the action of ribonuclease T1 combines with the action of the endogenous nuclease which makes the degradation process more difficult to analyze.