SPECIFIC VALYLATION OF TURNIP YELLOW MOSAIC-VIRUS RNA BY WHEAT-GERM VALYL-TRANSFER RNA-SYNTHETASE DETERMINED BY 3 ANTICODON LOOP NUCLEOTIDES
SPECIFIC VALYLATION OF TURNIP YELLOW MOSAIC-VIRUS RNA BY WHEAT-GERM VALYL-TRANSFER RNA-SYNTHETASE DETERMINED BY 3 ANTICODON LOOP NUCLEOTIDES
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DOI：
10.1021/bi00153a010
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发表时间：
1992-09-29
期刊：
影响因子：
2.9
通讯作者：
GIEGE, R
中科院分区：
文献类型：
--
作者：
DREHER, TW;TSAI, CH;GIEGE, R
研究了萝卜黄花叶病毒RNA反密码子环突变体在小麦胚谷氨酸- trna合成酶作用下的谷化作用。利用T7 RNA聚合酶从基因组RNA的3' trna样区cDNA中合成264个核苷酸长的RNA底物。反密码子环中三个核苷酸的单独或组合替换导致非常差的酰化(相对于野生型，V(max)/K（M)小于10(-3)）。因此，这些核苷酸代表了小麦胚芽缬氨酸trna合成酶识别的主要缬氨酸身份决定因素；它们对缬氨酸同源性的相对贡献由大到小依次为：反密码子的中间核苷酸（在TYMV RNA中为A56）、3‘反密码子核苷酸（C55）和最3’反密码子环核苷酸（C53）。振荡位置（C57）的取代对酰化动力学没有显著影响，而鉴别碱基（A4）的取代导致V(max)/K(m)的小幅下降。主要同源核苷酸的突变导致K(M)的大量增加，这表明小麦胚芽valyl-tRNA合成酶对低缬氨酸同源的变异底物具有较低的亲和力。与其他利用大肠杆菌和酵母的缬氨酸trna合成酶进行的研究比较表明，反密码子作为缬氨酸身份的优势区域在系统进化上是保守的，而鉴别碱基的身份贡献则不那么保守。反密码子突变的区分机制似乎也各不相同，小麦胚芽酶是基于亲和的，酵母酶是基于动力学的[Florentz et al. (1991) Eur。[j].生物化学学报，2004,22(2):444 - 444。
The valylation by wheat germ valyl-tRNA synthetase of anticodon loop mutants of turnip yellow mosaic virus RNA has been studied. RNA substrates 264 nucleotides long were made by T7 RNA polymerase from cDNA encompassing the 3' tRNA-like region of genomic RNA. Substitution singly, or in combination, of three nucleotides in the anticodon loop resulted in very poor valylation (V(max)/K(M) less than 10(-3) relative to wild type). These nucleotides thus represent the major valine identity determinants recognized by wheat germ valyl-tRNA synthetase; their relative contribution to valine identity, in descending order, was as follows: the middle nucleotide of the anticodon (A56 in TYMV RNA), the 3' anticodon nucleotide (C55), and the 3'-most anticodon loop nucleotide (C53). Substitutions in the wobble position (C57) had no significant effect on valylation kinetics, while substitutions of the discriminator base (A4) resulted in small decreases in V(max)/K(m). Mutations in the major identity nucleotides resulted in large increases in K(M), suggesting that wheat germ valyl-tRNA synthetase has a lowered affinity for variant substrates with low valine identity. Comparison with other studies using valyl-tRNA synthetases from Escherichia coli and yeast indicates that the anticodon has been phylogenetically conserved as the dominant valine identity region, while the identity contribution of the discriminator base has been less conserved. The mechanism by which anticodon mutations are discriminated also appears to vary, being affinity-based for the wheat germ enzyme, and kinetically-based for the yeast enzyme [Florentz et al. (1991) Eur. J. Biochem. 195, 229-234].