Bullock Timothy L, Rodríguez-Hernández Annia, Corigliano Eleonora M, Perona John J
Department of Chemistry and Biochemistry, and Interdepartmental Program in Biomolecular Science and Engineering, University of California, Santa Barbara, CA 93106-9510, USA.
Proc Natl Acad Sci U S A. 2008 May 27;105(21):7428-33. doi: 10.1073/pnas.0711812105. Epub 2008 May 13.
Information transfer from nucleic acid to protein is mediated by aminoacyl-tRNA synthetases, which catalyze the specific pairings of amino acids with transfer RNAs. Despite copious sequence and structural information on the 22 tRNA synthetase families, little is known of the enzyme signatures that specify amino acid selectivities. Here, we show that transplanting a conserved arginine residue from glutamyl-tRNA synthetase (GluRS) to glutaminyl-tRNA synthetase (GlnRS) improves the K(M) of GlnRS for noncognate glutamate. Two crystal structures of this C229R GlnRS mutant reveal that a conserved twin-arginine GluRS amino acid identity signature cannot be incorporated into GlnRS without disrupting surrounding protein structural elements that interact with the tRNA. Consistent with these findings, we show that cumulative replacement of other primary binding site residues in GlnRS, with those of GluRS, only slightly improves the ability of the GlnRS active site to accommodate glutamate. However, introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in C229R, improves the capacity of Escherichia coli GlnRS to synthesize misacylated Glu-tRNA(Gln) by 16,000-fold. This hybrid enzyme recapitulates the function of misacylating GluRS enzymes found in organisms that synthesize Gln-tRNA(Gln) by an alternative pathway. These findings implicate the RNA component of the contemporary GlnRS-tRNA(Gln) complex in mediating amino acid specificity. This role for tRNA may persist as a relic of primordial cells in which the evolution of the genetic code was driven by RNA-catalyzed amino acid-RNA pairing.
从核酸到蛋白质的信息传递由氨酰 - tRNA合成酶介导，该酶催化氨基酸与转运RNA的特异性配对。尽管有关于22个tRNA合成酶家族丰富的序列和结构信息，但对于决定氨基酸选择性的酶特征却知之甚少。在这里，我们表明，将谷氨酰胺 - tRNA合成酶（GlnRS）中的一个保守精氨酸残基移植到谷氨酰胺 - tRNA合成酶（GlnRS）中，可提高GlnRS对非同源谷氨酸的K(M)值。这种C229R GlnRS突变体的两个晶体结构表明，保守的双精氨酸GluRS氨基酸识别特征无法整合到GlnRS中，而不破坏与tRNA相互作用的周围蛋白质结构元件。与这些发现一致，我们表明，用GluRS的其他主要结合位点残基累积替换GlnRS中的残基，只会略微提高GlnRS活性位点容纳谷氨酸的能力。然而，引入22个氨基酸替换和一个缺失，包括替换整个主要结合位点和与C229R中破坏区域相邻的两个表面环，可将大肠杆菌GlnRS合成错误酰化的Glu - tRNA(Gln)的能力提高16000倍。这种杂合酶概括了通过替代途径合成Gln - tRNA(Gln)的生物体中发现的错误酰化GluRS酶的功能。这些发现暗示了当代GlnRS - tRNA(Gln)复合物的RNA成分在介导氨基酸特异性方面的作用。tRNA的这种作用可能作为原始细胞的遗迹而持续存在，在原始细胞中，遗传密码的进化是由RNA催化的氨基酸 - RNA配对驱动的。
