Schulman L H, Valenzuela D, Pelka H
Biochemistry. 1981 Oct 13;20(21):6018-23. doi: 10.1021/bi00524a015.
Protein affinity labeling groups have been attached to single-stranded cytidine residues in four structural regions of tRNAfMet. Modification of the tRNA with an average of one cross-linking group per molecule is achieved with retention of 75% of the original methionine acceptor activity. Incubation of the modified tRNA with methionyl-tRNA synthetase (MetRS) results in covalent coupling of the protein and nucleic acid by reaction of N-hydroxysuccinimide ester groups attached to the tRNA with lysine residues in the enzyme. In the presence of excess MetRS, approximately 30% of the input tRNA can be covalently bound to protein, indicating that lysine residues are appropriately oriented for reaction with cross-linking groups attached to certain sites in the tRNA but not to others. The cross-linking reaction results in loss of aminoacylation activity of MetRS equal to the amount of covalently bound tRNA. Enzyme activity is restored by release of bound tRNA following cleavage of the disulfide bond of the cross-linker with a sulfhydryl reagent. The data indicate that cross-linking occurs at the tRNA binding site of the enzyme. In the presence of excess modified tRNAfMet, a maximum of 1 mol of tRNA is cross-linked per mol of MetRS, in keeping with the known anticooperative tRNA binding properties of the native dimeric synthetase. In addition, the coupling reaction is effectively inhibited by unmodified tRNAfMet, but not by noncognate tRNAs.
蛋白质亲和标记基团已连接到甲硫氨酸起始转运核糖核酸（tRNAfMet）四个结构区域的单链胞苷残基上。通过每个分子平均连接一个交联基团对tRNA进行修饰，同时保留了75%的原始甲硫氨酸接受活性。将修饰后的tRNA与甲硫氨酰 - tRNA合成酶（MetRS）一起温育，会使tRNA上连接的N - 羟基琥珀酰亚胺酯基团与酶中的赖氨酸残基反应，从而导致蛋白质与核酸发生共价偶联。在过量MetRS存在的情况下，大约30%的输入tRNA可以与蛋白质共价结合，这表明赖氨酸残基的取向适合与连接在tRNA某些位点而非其他位点的交联基团发生反应。交联反应导致MetRS的氨酰化活性丧失，其丧失程度与共价结合的tRNA量相等。用巯基试剂切断交联剂的二硫键后，释放结合的tRNA可恢复酶活性。数据表明交联发生在酶的tRNA结合位点。在过量修饰的tRNAfMet存在的情况下，每摩尔MetRS最多交联1摩尔tRNA，这与天然二聚体合成酶已知的反协同tRNA结合特性一致。此外，未修饰的tRNAfMet可有效抑制偶联反应，但非同源tRNA则不能。
