浙江大学学报（理学版）
浙江大學學報（理學版）
절강대학학보（이학판）
JOURNAL OF ZHEJIANG UNIVERSITY
2009年
6期
708-713
,共6页
种属特异性元件%水稻线粒体tRNATrp%突变体%色氨酰 tRNA 合成酶
種屬特異性元件%水稻線粒體tRNATrp%突變體%色氨酰 tRNA 閤成酶
충속특이성원건%수도선립체tRNATrp%돌변체%색안선 tRNA 합성매
species-specific elements Oryza sativa mitochondria tRNA~(Trp)%mutant%tryptophanyl-tRNA synthetase (TrpRS)
为了研究水稻线粒体tRNA~(Trp)的种属特异性元件,在野生型水稻线粒体tRNA~(Trp)的基础上,设计并完成了3种向人tRNA~(Trp)的突变,体外转录并用枯草杆菌和人这两种不同种属来源的色氨酰-tRNA合成酶(TrpRS)测定了这些 tRNA~(Trp) 分子的氨酰化活力(K_(cat)/K_M).结果表明,与野生型水稻线粒体tRNA~(Trp)相比, 3个突变体被人TrpRS氨酰化的活力分别提高了354、407和803倍,其中以PMPH3(水稻线粒体tRNA~(Trp)的氨基酸接受茎的C2-G71和G3-C70都突变为人tRNA~(Trp)的氨基酸接受茎的相应部位)的氨酰化活力改变最大.而3个突变体对B.subtilis TrpRS氨酰化活力有进一步负影响,氨酰化活力微弱.说明水稻线粒体tRNA~(Trp)氨基酸接受茎上的第2个碱基对C2-G71和第3个碱基对G3-C70在人色氨酰-tRNA合成酶识别过程中有着极为重要的作用,是水稻线粒体tRNA~(Trp)的种属特异性元件.
為瞭研究水稻線粒體tRNA~(Trp)的種屬特異性元件,在野生型水稻線粒體tRNA~(Trp)的基礎上,設計併完成瞭3種嚮人tRNA~(Trp)的突變,體外轉錄併用枯草桿菌和人這兩種不同種屬來源的色氨酰-tRNA閤成酶(TrpRS)測定瞭這些 tRNA~(Trp) 分子的氨酰化活力(K_(cat)/K_M).結果錶明,與野生型水稻線粒體tRNA~(Trp)相比, 3箇突變體被人TrpRS氨酰化的活力分彆提高瞭354、407和803倍,其中以PMPH3(水稻線粒體tRNA~(Trp)的氨基痠接受莖的C2-G71和G3-C70都突變為人tRNA~(Trp)的氨基痠接受莖的相應部位)的氨酰化活力改變最大.而3箇突變體對B.subtilis TrpRS氨酰化活力有進一步負影響,氨酰化活力微弱.說明水稻線粒體tRNA~(Trp)氨基痠接受莖上的第2箇堿基對C2-G71和第3箇堿基對G3-C70在人色氨酰-tRNA閤成酶識彆過程中有著極為重要的作用,是水稻線粒體tRNA~(Trp)的種屬特異性元件.
위료연구수도선립체tRNA~(Trp)적충속특이성원건,재야생형수도선립체tRNA~(Trp)적기출상,설계병완성료3충향인tRNA~(Trp)적돌변,체외전록병용고초간균화인저량충불동충속래원적색안선-tRNA합성매(TrpRS)측정료저사 tRNA~(Trp) 분자적안선화활력(K_(cat)/K_M).결과표명,여야생형수도선립체tRNA~(Trp)상비, 3개돌변체피인TrpRS안선화적활력분별제고료354、407화803배,기중이PMPH3(수도선립체tRNA~(Trp)적안기산접수경적C2-G71화G3-C70도돌변위인tRNA~(Trp)적안기산접수경적상응부위)적안선화활력개변최대.이3개돌변체대B.subtilis TrpRS안선화활력유진일보부영향,안선화활력미약.설명수도선립체tRNA~(Trp)안기산접수경상적제2개감기대C2-G71화제3개감기대G3-C70재인색안선-tRNA합성매식별과정중유착겁위중요적작용,시수도선립체tRNA~(Trp)적충속특이성원건.
To discover the species-specific element of Oryza sativa mitochondria tRNA~(Trp),three mutants from Oryza sativa mitochondria tRNA~(Trp) to human tRNA~(Trp) (PMPH1: 2-71 (CA→ GU) ; PMPH2: 3-70(GC→CG) ; PMPH3: 2-71(CA→GU) and 3-70 (GC→CG) were constructed and transcribed in vitro with T7 RNA polymerase.The kinetic parameters (K_(cat),/K_M) of B.subtilis tryptophanyl-tRNA synthetase (TrpRS)and human TrpRS were determined with three mutant-type tRNA~(Trp).The results showed that the aminoacryl activities of these three mutants were 354,407,and 803 times higher than the wild-type Oryza sativa mitochondria tRNA~(Trp).The PMPH3 based on Oryza sativa mitochondrial tRNA~(Trp) with its C2/G71and G3/C70 bases were transplanted by Human counterparts showed a large increase in aminoacylation efficiency by human TrpRS.The three mutants all showed a weak amino-acylation efficiency by B.subtilis TrpRS as compared with the wild-type Oryza sativa Mitochondrial tRNA~(Trp).It was concluded that the bases of C2-G71 and G3-C70 play a very important role in recognition by human TrpRS and is the species-specific elements in Oryza sativa mitochondria tRNA~(Trp).
