Stuart J W, Gdaniec Z, Guenther R, Marszalek M, Sochacka E, Malkiewicz A, Agris P F
Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622, USA.
Biochemistry. 2000 Nov 7;39(44):13396-404. doi: 10.1021/bi0013039.
The structure of the human tRNA(Lys3) anticodon stem and loop domain (ASL(Lys3)) provides evidence of the physicochemical contributions of N6-threonylcarbamoyladenosine (t(6)A(37)) to tRNA(Lys3) functions. The t(6)A(37)-modified anticodon stem and loop domain of tRNA(Lys3)(UUU) (ASL(Lys3)(UUU)- t(6)A(37)) with a UUU anticodon is bound by the appropriately programmed ribosomes, but the unmodified ASL(Lys3)(UUU) is not [Yarian, C., Marszalek, M., Sochacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., and Agris, P. F., Biochemistry 39, 13390-13395]. The structure, determined to an average rmsd of 1.57 +/- 0.33 A (relative to the mean structure) by NMR spectroscopy and restrained molecular dynamics, is the first reported of an RNA in which a naturally occurring hypermodified nucleoside was introduced by automated chemical synthesis. The ASL(Lys3)(UUU)-t(6)A(37) loop is significantly different than that of the unmodified ASL(Lys3)(UUU), although the five canonical base pairs of both ASL(Lys3)(UUU) stems are in the standard A-form of helical RNA. t(6)A(37), 3'-adjacent to the anticodon, adopts the form of a tricyclic nucleoside with an intraresidue H-bond and enhances base stacking on the 3'-side of the anticodon loop. Critically important to ribosome binding, incorporation of the modification negates formation of an intraloop U(33).A(37) base pair that is observed in the unmodified ASL(Lys3)(UUU). The anticodon wobble position U(34) nucleobase in ASL(Lys3)(UUU)-t(6)A(37) is significantly displaced from its position in the unmodified ASL and directed away from the codon-binding face of the loop resulting in only two anticodon bases for codon binding. This conformation is one explanation for ASL(Lys3)(UUU) tendency to prematurely terminate translation and -1 frame shift. At the pH 5.6 conditions of our structure determination, A(38) is protonated and positively charged in ASL(Lys3)(UUU)-t(6)A(37) and the unmodified ASL(Lys3)(UUU). The ionized carboxylic acid moiety of t(6)A(37) possibly neutralizes the positive charge of A(+)(38). The protonated A(+)(38) can base pair with C(32), but t(6)A(37) may weaken the interaction through steric interference. From these results, we conclude that ribosome binding cannot simply be an induced fit of the anticodon stem and loop, otherwise the unmodified ASL(Lys3)(UUU) would bind as well as ASL(Lys3)(UUU)-t(6)A(37). t(6)A(37) and other position 37 modifications produce the open, structured loop required for ribosomal binding.
人tRNA（Lys3）反密码子茎环结构域（ASL（Lys3））的结构为N6-苏氨甲酰腺苷（t（6）A（37））对tRNA（Lys3）功能的物理化学贡献提供了证据。具有反密码子UUU的tRNA（Lys3）（UUU）的t（6）A（37）修饰的反密码子茎环结构域（ASL（Lys3）（UUU）-t（6）A（37））被适当编程的核糖体结合，但未修饰的ASL（Lys3）（UUU）则不然[Yarian, C., Marszalek, M., Sochacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., and Agris, P. F., Biochemistry 39, 13390 - 13395]。通过核磁共振光谱和受限分子动力学确定的平均均方根偏差为1.57±0.33 Å（相对于平均结构）的结构，是首次报道的通过自动化学合成引入天然存在超修饰核苷的RNA结构。ASL（Lys3）（UUU）-t（6）A（37）环与未修饰的ASL（Lys3）（UUU）环有显著差异，尽管两个ASL（Lys3）（UUU）茎的五个经典碱基对均处于螺旋RNA的标准A构象。位于反密码子3'相邻位置的t（6）A（37）采用具有残基内氢键的三环核苷形式，并增强了反密码子环3'侧的碱基堆积。对核糖体结合至关重要的是，该修饰的掺入消除了未修饰的ASL（Lys3）（UUU）中观察到的环内U（33）.A（37）碱基对的形成。ASL（Lys3）（UUU）-t（6）A（37）中的反密码子摆动位置U（34）核苷酸碱基与其在未修饰的ASL中的位置有显著位移，并远离环的密码子结合面，导致只有两个反密码子碱基用于密码子结合。这种构象是ASL（Lys3）（UUU）倾向于过早终止翻译和 - 1移码的一种解释。在我们确定结构的pH 5.6条件下，ASL（Lys3）（UUU）-t（6）A（37）和未修饰的ASL（Lys3）（UUU）中的A（38）被质子化并带正电荷。t（6）A（37）的离子化羧酸部分可能中和A（+）（38）的正电荷。质子化的A（+）（38）可以与C（32）形成碱基对，但t（6）A（37）可能通过空间位阻削弱这种相互作用。从这些结果我们得出结论，核糖体结合不能简单地是反密码子茎环的诱导契合，否则未修饰的ASL（Lys3）（UUU）也会像ASL（Lys3）（UUU）-t（6）A（37）一样结合。t（6）A（37）和其他37位修饰产生了核糖体结合所需的开放、结构化环。
