Structural and functional roles of the N1-and N3-protons of Ψ at tRNA's position 39
Structural and functional roles of the N1-and N3-protons of Ψ at tRNA's position 39
复制标题
DOI：
10.1093/nar/27.17.3543
复制
发表时间：
1999-09-01
影响因子：
14.9
通讯作者：
Agris, PF
中科院分区：
文献类型：
--
作者：
Yarian, CS;Basti, MM;Agris, PF
tRNA 反密码子茎环结构域 (ASL) 39 位的伪尿苷 (Psi(39)) 高度保守。为了确定 Psi(39) 对 ASL 的物理化学贡献并将这些特性与 tRNA 翻译功能联系起来，我们合成了未修饰的酵母 tRNA(Phe) ASL 和具有 U-39 和 Psi(39) 的各种衍生物的 ASL。与未修饰的 ASL (K-d = 197 +/- 58 nM) 相比，Psi(39) 增加了 ASL 的热稳定性 (Delta T-m = 1.3 +/- 0.5 摄氏度)，但没有显着影响核糖体结合 (K-d = 229 +/- 29 nM)。 30S核糖体亚基上的ASL-Psi(39) P位点指纹与未修饰的ASL相似，具有m(1)Psi(39)的ASL的稳定性、核糖体结合和指纹与具有Psi(39)的ASL相当。因此，Psi(39) 对 ASL 稳定性的贡献与 N1-H 氢键无关，但可能是由于与 U 相比，核苷能够改善碱基堆积。相反，m(3)Psi(39)、等排 m(3)U(39) 和 m(1)m(3)Psi(39) 的取代通过破坏 A(31)-U-39 碱基对来破坏 ASL 的稳定性。核磁共振证实，茎中 U 和 Psi 的 N3 甲基化显着降低了核糖体结合（K-d = 1060 +/- 189 至 1283 +/- 258 nM）。因此，Psi(39) 通过 N3-H 与 A(31) 的规范碱基配对对于结构、稳定性和核糖体结合非常重要，而 U-39 修饰为 Psi(39) 所提供的稳定性增加和 N1-质子可能具有除 tRNA 与核糖体 P 位点结合以外的生物学作用。
Pseudouridine at position 39 (Psi(39)) of tRNA's anticodon stem and loop domain (ASL) is highly conserved. To determine the physicochemical contributions of Psi(39) to the ASL and to relate these properties to tRNA function in translation, we synthesized the unmodified yeast tRNA(Phe) ASL and ASLs with various derivatives of U-39 and Psi(39). Psi(39) increased the thermal stability of the ASL (Delta T-m = 1.3 +/- 0.5 degrees C), but did not significantly affect ribosomal binding (K-d = 229 +/- 29 nM) compared to that of the unmodified ASL (K-d = 197 +/- 58 nM). The ASL-Psi(39) P-site fingerprint on the 30S ribosomal subunit was similar to that of the unmodified ASL, The stability, ribosome binding and fingerprint of the ASL with m(1)Psi(39) were comparable to that of the ASL with Psi(39). Thus, the contribution of Psi(39) to ASL stability is not related to N1-H hydrogen bonding, but probably is due to the nucleoside's ability to improve base stacking compared to U. In contrast, substitutions of m(3)Psi(39), the isosteric m(3)U(39) and m(1)m(3)Psi(39) destabilized the ASL by disrupting the A(31)-U-39 base pair in the stem, as confirmed by NMR, N3-methylations of both U and Psi dramatically decreased ribosomal binding (K-d = 1060 +/- 189 to 1283 +/- 258 nM). Thus, canonical base pairing of Psi(39) to A(31) through N3-H is important to structure, stability and ribosome binding, whereas the increased stability and the N1-proton afforded by modification of U-39 to Psi(39) may have biological roles other than tRNA's binding to the ribosomal P-site.