Alexander R W, Nordin B E, Schimmel P
The Skaggs Institute for Chemical Biology, The Scripps Research Institute, Beckman Center, 10560 North Torrey Pines Road, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12214-9. doi: 10.1073/pnas.95.21.12214.
We report that aminoacylation of minimal RNA helical substrates is enhanced by mismatched or unpaired nucleotides at the first position in the helix. Previously, we demonstrated that the class I methionyl-tRNA synthetase aminoacylates RNA microhelices based on the acceptor stem of initiator and elongator tRNAs with greatly reduced efficiency relative to full-length tRNA substrates. The cocrystal structure of the class I glutaminyl-tRNA synthetase with tRNAGln revealed an uncoupling of the first (1.72) base pair of tRNAGln, and tRNAMet was proposed by others to have a similar base-pair uncoupling when bound to methionyl-tRNA synthetase. Because the anticodon is important for efficient charging of methionine tRNA, we thought that 1.72 distortion is probably effected by the synthetase-anticodon interaction. Small RNA substrates (minihelices, microhelices, and duplexes) are devoid of the anticodon triplet and may, therefore, be inefficiently aminoacylated because of a lack of anticodon-triggered acceptor stem distortion. To test this hypothesis, we constructed microhelices that vary in their ability to form a 1.72 base pair. The results of kinetic assays show that microhelix aminoacylation is activated by destabilization of this terminal base pair. The largest effect is seen when one of the two nucleotides of the pair is completely deleted. Activation of aminoacylation is also seen with the analogous deletion in a minihelix substrate for the closely related isoleucine enzyme. Thus, for at least the methionine and isoleucine systems, a built-in helix destabilization compensates in part for the lack of presumptive anticodon-induced acceptor stem distortion.
我们报告称，在螺旋结构的第一个位置上存在错配或未配对核苷酸时，最小RNA螺旋底物的氨酰化作用会增强。此前，我们证明，I类甲硫氨酰 - tRNA合成酶对基于起始tRNA和延伸tRNA受体茎的RNA微螺旋进行氨酰化的效率，相对于全长tRNA底物大幅降低。I类谷氨酰胺 - tRNA合成酶与tRNAGln的共晶体结构揭示了tRNAGln的第一个（1.72）碱基对解偶联，其他人提出tRNAMet与甲硫氨酰 - tRNA合成酶结合时也有类似的碱基对解偶联。由于反密码子对于甲硫氨酸tRNA的有效充电很重要，我们认为1.72扭曲可能是由合成酶 - 反密码子相互作用引起的。小RNA底物（微螺旋、微螺旋和双链体）缺乏反密码子三联体，因此可能由于缺乏反密码子触发的受体茎扭曲而氨酰化效率低下。为了验证这一假设，我们构建了在形成1.72碱基对能力上有所不同的微螺旋。动力学测定结果表明，微螺旋氨酰化通过该末端碱基对的去稳定化而被激活。当该碱基对的两个核苷酸之一完全缺失时，效果最为明显。在与异亮氨酸酶密切相关的微螺旋底物中进行类似缺失时，也观察到了氨酰化的激活。因此，至少对于甲硫氨酸和异亮氨酸系统，内在的螺旋去稳定化部分补偿了假定的反密码子诱导的受体茎扭曲的缺乏。
