Horowitz J, Chu W C, Derrick W B, Liu J C, Liu M, Yue D
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames 50011, USA.
Biochemistry. 1999 Jun 15;38(24):7737-46. doi: 10.1021/bi990490b.
We have studied the interactions between Escherichia coli tRNAVal and valyl-tRNA synthetase (ValRS) by enzymatic footprinting with nuclease S1 and ribonuclease V1, and by analysis of the aminoacylation kinetics of mutant tRNAVal transcripts. Valyl-tRNA synthetase specifically protects the anticodon loop, the 3' side of the stacked T-stem/acceptor-stem helix, and the 5' side of the anticodon stem of tRNAVal against cleavage by double- and single-strand-specific nucleases. Increased nuclease susceptibility at the ends of the anticodon- and T-stems in the tRNAVal.ValRS complex is indicative of enzyme-induced conformational changes in the tRNA. The most important synthetase recognition determinants are the middle and 3' anticodon nucleotides (A35 and C36, respectively); G20, in the variable pocket, and G45, in the tRNA central core, are minor recognition elements. The discriminator base, position 73, and the anticodon stem also are recognized by ValRS. Replacing wild-type A73 with G73 reduces the aminoacylation efficiency more than 40-fold. However, the C73 and U73 mutants remain good substrates for ValRS, suggesting that guanosine at position 73 acts as a negative determinant. The amino acid acceptor arm of tRNAVal contains no other synthetase recognition nucleotides, but regular A-type RNA helix geometry in the acceptor stem is essential [Liu, M., et al. (1997) Nucleic Acids Res. 25, 4883-4890]. In the anticodon stem, converting the U29:A41 base pair to C29:G41 reduces the aminoacylation efficiency 50-fold. This is apparently due to the rigidity of the anticodon stem caused by the presence of five consecutive C:G base pairs, since the A29:U41 mutant is readily aminoacylated. Identity switch experiments provide additional evidence for a role of the anticodon stem in synthetase recognition. The valine recognition determinants, A35, C36, A73, G20, G45, and a regular A-RNA acceptor helix are insufficient to transform E. coli tRNAPhe into an effective valine acceptor. Replacing the anticodon stem of tRNAPhe with that of tRNAVal, however, converts the tRNA into a good substrate for ValRS. These experiments confirm G45 as a minor ValRS recognition element.
我们通过核酸酶S1和核糖核酸酶V1的酶足迹法以及对突变型tRNAVal转录本的氨酰化动力学分析，研究了大肠杆菌tRNAVal与缬氨酰 - tRNA合成酶（ValRS）之间的相互作用。缬氨酰 - tRNA合成酶能特异性保护tRNAVal的反密码子环、堆积的T - 茎/受体茎螺旋的3'侧以及反密码子茎的5'侧，使其免受双链和单链特异性核酸酶的切割。tRNAVal - ValRS复合物中反密码子茎和T - 茎末端核酸酶敏感性增加，表明酶诱导了tRNA的构象变化。最重要的合成酶识别决定因素是反密码子的中间和3'核苷酸（分别为A35和C36）；可变口袋中的G20和tRNA中心核心中的G45是次要识别元件。ValRS也能识别判别碱基（第73位）和反密码子茎。将野生型A73替换为G73会使氨酰化效率降低40多倍。然而，C73和U73突变体仍然是ValRS的良好底物，这表明第73位的鸟苷作为一个负决定因素。tRNAVal的氨基酸接受臂不包含其他合成酶识别核苷酸，但接受茎中规则的A型RNA螺旋结构是必不可少的[Liu, M., 等人（1997年）《核酸研究》25, 4883 - 4890]。在反密码子茎中，将U29:A41碱基对转换为C29:G41会使氨酰化效率降低50倍。这显然是由于五个连续的C:G碱基对导致反密码子茎的刚性增加，因为A29:U41突变体很容易被氨酰化。身份转换实验为反密码子茎在合成酶识别中的作用提供了额外证据。缬氨酸识别决定因素A35、C36、A73、G20、G45和规则的A - RNA接受螺旋不足以将大肠杆菌tRNAPhe转化为有效的缬氨酸接受体。然而，用tRNAVal的反密码子茎替换tRNAPhe的反密码子茎，会使该tRNA成为ValRS的良好底物。这些实验证实G45是一个次要的ValRS识别元件。
