Functional Structure of the GTPase Domain within 28S Ribosomal RNA (Analysis of a Conformational Epitope on RNA Recognized by a Unique Autoantibody)

核糖体RNA是高度保守的大分子。目前的证据表明rRNA在蛋白质生物合成中的功能作用。然而，对于rRNA在蛋白质合成过程中的详细功能结构及其作用机制，我们几乎一无所知。1991年，我们在自身免疫性疾病患者的血清中发现了一种位点特异性抗rrna自身抗体（抗28s）。抗- 28s强烈抑制延伸因子与核糖体相互作用的蛋白合成。在1992 - 1994年的三年时间里，我们以anti-28为探针，对rRNA抗体结合区的结构和功能进行了研究。我们报道了以下发现并阐明了RNA结构域与延伸因子和相关GTP水解的相互作用：(1)Anti-28S特异性地保护28S rRNA中称为“GTPase结构域”的区域免受RNase酶切和化学修饰。包括G-1959在内的四个碱基被确定为参与自身免疫表位形成的元件。(2)核糖体蛋白P1、P2、P0和L12结合GPTase结构域，影响RNA结构域的三级结构。(3)原核核糖体对anti-28S的可及性很低。然而，a -1067碱基置换到G的位置相当于真核1959位点，导致核糖体的抗28s可及性高，表明G-1959对ani - 28s识别很重要。(4) G-1959还受到延伸因子EF-2的保护，不受硫酸二甲基1的化学修饰，这表明抗28s直接阻断了EF-2对GTPase结构域的作用。(5) 17% SLE患者血清中检测到抗28s活性。所有抗28s血清还含有与该结构域相关蛋白P1、P2和P0反应的抗p抗体。

Ribosomal RNA (rRNA) is highly conserved macromolecule. Currnet evidence shows a functional role of rRNA in protein biosynthesis. However, almost nothing is known about the detailed functional structure of the rRNA and mechanism of its action in the process of protein synthesis. In 1991, we found a site-specific anti-rRNA autoantibody (anti-28S) in sera from patients with autoimmune disease. Anti-28S strongly inhibits protein synthesis at the step of interaction between elongation factors and ribosomes. In this project during three years, 1992 to 1994, we have studied on the structure and function of the antibody-binding region of rRNA using anti-28as a probe. We have reported following findings and clarified involvement of the RNA domain in interaction with elongation factors and the associated GTP hydrolysis :(1) Anti-28S specifically protects a region termed the "GTPase domain" within 28S rRNA from RNase digestion and chemical modifications. Four bases including G-1959 were identified as elements involved in formation of the autoimmune epitope.(2) Ribosomal proteins P1, P2, P0, and L12 bind to be GPTase domain and affect the tertiary structure of the RNA domain.(3) Prokaryotic ribosomes shows very low accessibility to anti-28S.However, a base substitution of A-1067 to G at the position equivalent to eukaryotic 1959 site causes high anti-28S accessibility of the ribosomes, indicating G-1959 is important for ansi-28S recognition.(4) G-1959 is also protected by elongation factor EF-2 from chmical modification with dimethy1 sulfate, suggesting that anti-28S directly blocks the EF-2 action to the GTPase domain.(5) Anti-28S activity is detected in sera from 17 % patients with SLE.All the anti-28S sera also contain anti-P antibody reactive with tha domain-associating proteins P1, P2, and P0.