Wickstrom E, Behlen L S, Reuben M A, Ainpour P R
Proc Natl Acad Sci U S A. 1981 Apr;78(4):2082-5. doi: 10.1073/pnas.78.4.2082.
Intramolecular crosslinking of yeast phenylalanine tRNA in aqueous solution with rigid, variable-length crosslinking reagents, which we call "molecular rulers," has given results in reasonable agreement with the crystal structure. Chlorambucilyl-[3H]phenylalanyl-tRNAPhe crosslinked intramolecularly at G-71 and A-73, whereas chlorambucilyl-pentadecaprolyl-[3H]phenylalanyl-tRNAPhe crosslinked at G-20 and Y-37. The pentadecaprolyl reagent was predicted to be 62 A long, including chlorambucil and phenylalanine; the sites that it reached are 60 A distant from the 3' OH (in the case of G-20) or 80 A distant (in the case of Y-37) in the crystal structure of tRNAPhe. The close agreement between the length of the reagent and the distance of G-20 from the 3' OH in the crystal structure illustrates the rigidity of the tRNAPhe molecule in the dihydrouridine loop region at the corner of the molecule. The apparent ability of the 62-A-long reagent to crosslink to a site, Y-37, that is 80 A distant from the 3' OH in the crystal structure appears to illustrate the flexibility of both the 3' A-C-C-A terminus and the anticodon stem and loop, with respect to the tRNA molecule. These observations demonstrate the utility of oligoproline-based crosslinking reagents as rigid, variable-length molecular rulers for biological macromolecules in solution.
在水溶液中，用我们称为“分子尺”的刚性、可变长度交联剂对酵母苯丙氨酸tRNA进行分子内交联，所得结果与晶体结构相当吻合。苯丁酸氮芥基-[³H]苯丙氨酰-tRNAPhe在G-71和A-73处发生分子内交联，而苯丁酸氮芥基-十五聚脯氨酰-[³H]苯丙氨酰-tRNAPhe在G-20和Y-37处发生交联。预测十五聚脯氨酰试剂长度为62埃，包括苯丁酸氮芥和苯丙氨酸；在苯丙氨酸tRNA的晶体结构中，它所交联的位点距离3' OH在G-20的情况下为60埃，在Y-37的情况下为80埃。试剂长度与晶体结构中G-段距离3' OH的距离之间的紧密吻合，说明了苯丙氨酸tRNA分子在分子角落的二氢尿嘧啶环区域的刚性。在晶体结构中，62埃长的试剂明显能够交联到距离3' OH 80埃的位点Y-37，这似乎说明了3' A-C-C-A末端以及反密码子茎和环相对于tRNA分子的灵活性。这些观察结果证明了基于寡聚脯氨酸的交联剂作为溶液中生物大分子的刚性、可变长度分子尺的实用性。
