Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.
PLoS One. 2007 Jun 13;2(6):e517. doi: 10.1371/journal.pone.0000517.
Decoding of mRNAs is performed by aminoacyl tRNAs (aa-tRNAs). This process is highly accurate, however, at low frequencies (10(-3) - 10(-4)) the wrong aa-tRNA can be selected, leading to incorporation of aberrant amino acids. Although our understanding of what constitutes the correct or cognate aa-tRNA:mRNA interaction is well defined, a functional distinction between near-cognate or single mismatched, and unpaired or non-cognate interactions is lacking.
METHODOLOGY/PRINCIPAL FINDINGS: Misreading of several synonymous codon substitutions at the catalytic site of firefly luciferase was assayed in Saccharomyces cerevisiae. Analysis of the results in the context of current kinetic and biophysical models of aa-tRNA selection suggests that the defining feature of near-cognate aa-tRNAs is their potential to form mini-helical structures with A-site codons, enabling stimulation of GTPase activity of eukaryotic Elongation Factor 1A (eEF1A). Paromomycin specifically stimulated misreading of near-cognate but not of non-cognate aa-tRNAs, providing a functional probe to distinguish between these two classes. Deletion of the accessory elongation factor eEF1Bgamma promoted increased misreading of near-cognate, but hyperaccurate reading of non-cognate codons, suggesting that this factor also has a role in tRNA discrimination. A mutant of eEF1Balpha, the nucleotide exchange factor for eEF1A, promoted a general increase in fidelity, suggesting that the decreased rates of elongation may provide more time for discrimination between aa-tRNAs. A mutant form of ribosomal protein L5 promoted hyperaccurate decoding of both types of codons, even though it is topologically distant from the decoding center.
CONCLUSIONS/SIGNIFICANCE: It is important to distinguish between near-cognate and non-cognate mRNA:tRNA interactions, because such a definition may be important for informing therapeutic strategies for suppressing these two different categories of mutations underlying many human diseases. This study suggests that the defining feature of near-cognate aa-tRNAs is their potential to form mini-helical structures with A-site codons in the ribosomal decoding center. An aminoglycoside and a ribosomal factor can be used to distinguish between near-cognate and non-cognate interactions.
mRNA 的解码由氨酰-tRNA（aa-tRNA）完成。这个过程非常精确，但在低频（10(-3) - 10(-4))时，错误的 aa-tRNA 可能被选择，导致异常氨基酸的掺入。尽管我们对正确或同源 aa-tRNA:mRNA 相互作用的构成有很好的了解，但对近同源或单个错配与未配对或非同源相互作用之间的功能区别缺乏了解。
方法/主要发现：在酿酒酵母中检测了萤火虫荧光素酶催化位点的几个同义密码子替换的错读。在当前 aa-tRNA 选择的动力学和生物物理模型的背景下分析结果表明，近同源 aa-tRNA 的定义特征是它们与 A 位密码子形成小螺旋结构的潜力，从而能够刺激真核延伸因子 1A（eEF1A）的 GTPase 活性。巴龙霉素特异性地刺激近同源但不刺激非同源 aa-tRNA 的错读，提供了一种区分这两种类型的功能探针。辅助延伸因子 eEF1Bgamma 的缺失促进了近同源错读的增加，但对非同源密码子的超精确阅读，表明该因子在 tRNA 鉴别中也有作用。eEF1A 的核苷酸交换因子 eEF1Balpha 的突变体促进了保真度的普遍提高，这表明延伸率的降低可能为 aa-tRNA 之间的鉴别提供更多时间。核糖体蛋白 L5 的突变体促进了两种类型的密码子的超精确解码，尽管它在拓扑上远离解码中心。
结论/意义：区分近同源和非同源 mRNA:tRNA 相互作用很重要，因为这种定义可能对告知抑制许多人类疾病中这两种不同类型突变的治疗策略很重要。本研究表明，近同源 aa-tRNA 的定义特征是它们与核糖体解码中心的 A 位密码子形成小螺旋结构的潜力。氨基糖苷类药物和核糖体因子可用于区分近同源和非同源相互作用。
