Marion M J, Marion C, Arpin M, Reboud A M, Reboud J P
Nucleic Acids Res. 1976 Oct;3(10):2563-73. doi: 10.1093/nar/3.10.2563.
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.
通过比较用核糖核酸酶T1处理紧密的、缺乏K+的60S亚基和经EDTA处理的60S亚基后获得的结果，研究了核糖体亚基内28S RNA对核糖核酸酶T1的可及性。使用十二烷基硫酸钠和苯酚的混合物从亚基中提取RNA，并在蔗糖梯度上进行分析。来自活性亚基的RNA仅在高酶浓度下被降解。在缺乏K+的亚基中，RNA更容易被接触到，因为它分解成6个明确的片段，沉降在4S和18.5S之间。在EDTA处理的亚基内，RNA不再受到保护。事实上，它像游离的28S RNA一样，在低酶浓度下就被降解，产生异质片段。亚基和游离28S RNA的解链曲线比较表明，只有在EDTA处理的亚基中，蛋白质才不会稳定RNA的二级结构。对于40S亚基，核糖核酸酶T1的作用与内源性核酸酶的作用相结合，这使得降解过程更难分析。
