Slobin L I
Proc Natl Acad Sci U S A. 1972 Dec;69(12):3769-73. doi: 10.1073/pnas.69.12.3769.
To test whether a 30S ribosomal subunitformylmethionyl-tRNA-mRNA complex is an obligatory intermediate in protein synthesis, 70S ribosomes from Escherichia coli were crosslinked with the bifunctional imidoester, dimethylsuberimidate. Crosslinked ribosomes contained covelently joined 30S and 50S subunits, as judged by their inability to dissociate at low Mg(2+) concentrations. Treatment of 70S ribosomes with high salt (1 M NH(4)Cl), either before or after reaction with the crosslinking reagent, produced two different crosslinked ribosomal particles, one of "60 S" and the other "70 S." Preliminary evidence indicates that both particles can bind N-acetylphenylalanyl-tRNA at low Mg(2+) concentrations and are active for polyphenylalanine syntheses. Crosslinked ribosomes were functional when tested with poly(U) as an mRNA in systems requiring initiation factors and N-acetylphenylalanyl-tRNA for activity. Under optimal crosslinking conditions, they retained 80% of the activity of unmodified ribosomes for polyphenylalanine synthesis. Despite the maintenance of these functional capacities, such ribosomes had a sharply reduced ability to bind fMet-tRNA and were completely inactive in protein synthesis with bacteriophage f2 RNA as a messenger. We conclude that 70S ribosomes must dissociate into subunits to initiate protein synthesis with natural mRNAs.
为了测试30S核糖体亚基甲酰甲硫氨酰 - tRNA - mRNA复合物是否是蛋白质合成中的一个必需中间体，用双功能亚胺酯——二甲基辛二亚胺处理来自大肠杆菌的70S核糖体。通过在低镁离子浓度下它们无法解离来判断，交联的核糖体含有共价连接的30S和50S亚基。在与交联剂反应之前或之后，用高盐（1M氯化铵）处理70S核糖体，产生了两种不同的交联核糖体颗粒，一种是“60S”，另一种是“70S”。初步证据表明，这两种颗粒在低镁离子浓度下都能结合N - 乙酰苯丙氨酰 - tRNA，并且对聚苯丙氨酸合成有活性。当在需要起始因子和N - 乙酰苯丙氨酰 - tRNA才能有活性的系统中用聚（U）作为mRNA进行测试时，交联的核糖体是有功能的。在最佳交联条件下，它们保留了未修饰核糖体80%的聚苯丙氨酸合成活性。尽管保持了这些功能能力，但这样的核糖体结合甲硫氨酰 - tRNA的能力急剧下降，并且在以噬菌体f2 RNA作为信使的蛋白质合成中完全无活性。我们得出结论，70S核糖体必须解离成亚基才能用天然mRNA起始蛋白质合成。
