A primer extension (toeprinting) assay was used to monitor selection by ribosomes of the first versus the second AUG codon as a function of introducing mutations on the 3' side (positions +4, +5 and +6) of the first AUG codon. Six different flanking codons starting with G (GCG, GCU, GCC, GCA, GAU and GGA) strongly augmented selection of AUG#1 when compared with matched mRNAs that had A or C instead of G in position +4. Augmentation by G in position +4 failed only when it was combined with U in position +5, as in the sequence augGUA. In contrast with the usual enhancing effect of introducing G in position +4, most mutations in position +5 had no discernible effect, as shown with the series augANA (where N = C, A, G or U) and the series augCNA. AUG codon recognition was also unaffected by mutations in position +6, as shown by testing four mRNAs that had augCCN as the start site. Thus the primary sequence context that augments the recognition of AUG start codons does not appear generally to extend beyond G in position +4. When the toeprinting assay was used with mRNAs that initiate translation at CUG instead of AUG, cugGAU was not recognized better than cugGGU, contradicting the hypothesis that initiation at non-AUG codons might be favored by A instead of G in position +5.

译文

引物延伸 (toeprinting) 测定法用于监测第一个AUG密码子与第二个AUG密码子的核糖体选择,这是在第一个AUG密码子的3' 侧 (位置4、5和6) 引入突变的函数。与在4位具有A或C而不是G的匹配mrna相比,以G开头的六个不同的侧翼密码子 (GCG,GCU,GCC,GCA,GAU和GGA) 强烈增强了AUG #1的选择。仅当G在位置4与U在位置5结合时,G的增强才失败,如序列augGUA所示。与通常在4位引入G的增强作用相反,5位的大多数突变没有明显的作用,如augANA系列 (其中N = C,A,G或U) 和augCNA系列所示。AUG密码子识别也不受6位突变的影响,如测试四个以augCCN为起始位点的mrna所示。因此,增强对AUG起始密码子的识别的主要序列上下文通常不会扩展到4位中的G以外。当toeprinting测定法与在CUG而不是AUG启动翻译的mrna一起使用时,cugGAU的识别并不比cugGGU更好,这与以下假设相矛盾: A而不是G在5位可能有利于非AUG密码子启动。

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