Musier-Forsyth K, Schimmel P
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1994 Jan 25;33(3):773-9. doi: 10.1021/bi00169a019.
The 875 amino acid class II Escherichia coli alanine tRNA synthetase aminoacylates hairpin minihelices and miniduplexes comprising complementary base pairs that reconstruct the acceptor helix of alanine tRNA. Aminoacylation is dependent upon a G3:U70 base pair in the tRNA acceptor stem. A synthetic RNA miniduplex with a phosphorothioate internucleotide linkage on the 5'-side of U70 facilitated the stable attachment of a pendant benzophenone to the ribonucleotide backbone. The benzophenone-labeled duplex is active for aminoacylation. Irradiation of the labeled duplex produced a cross-linked RNA protein complex, in which the major site of RNA attachment is the segment between the class II defining sequence motifs 2 and 3. This segment spans a putative zinc-binding motif, which has been implicated in acceptor helix recognition, and is within a 461 amino acid N-terminal fragment that was recently shown to have full activity for minihelix aminoacylation. These results, together with the X-ray crystallographic investigations of the class II aspartate tRNA synthetase-tRNA(Asp) complex, suggest that the segment between motifs 2 and 3 in the 10 class II synthetases contributes generally to the docking of tRNA acceptor helices. The sequence diversity of this segment implies that its mode of interaction with the acceptor helix is idiosyncratic to the class II enzyme.
含有875个氨基酸的II类大肠杆菌丙氨酸tRNA合成酶可将发夹状小螺旋和小双链体氨酰化，这些小螺旋和小双链体包含互补碱基对，可重建丙氨酸tRNA的受体螺旋。氨酰化作用依赖于tRNA受体茎中的G3:U70碱基对。一种在U70的5'侧带有硫代磷酸酯核苷酸间连接的合成RNA小双链体促进了一个悬挂的二苯甲酮与核糖核苷酸主链的稳定连接。用二苯甲酮标记的双链体对氨酰化有活性。对标记的双链体进行辐照产生了一种交联的RNA-蛋白质复合物，其中RNA附着的主要位点是II类定义序列基序2和3之间的片段。该片段跨越一个推测的锌结合基序，该基序与受体螺旋识别有关，并且位于一个461个氨基酸的N端片段内，最近显示该片段对小螺旋氨酰化具有完全活性。这些结果，连同对II类天冬氨酸tRNA合成酶-tRNA(Asp)复合物的X射线晶体学研究，表明10种II类合成酶中基序2和3之间的片段通常有助于tRNA受体螺旋的对接。该片段的序列多样性意味着其与受体螺旋的相互作用模式对II类酶而言是特异的。
