Jackson R J
Department of Biochemistry, University of Cambridge, England.
Eur J Biochem. 1991 Sep 1;200(2):285-94. doi: 10.1111/j.1432-1033.1991.tb16184.x.
The requirement for ATP for initiation of eukaryotic mRNA translation was tested using gel-filtered rabbit reticulocyte lysates incubated with labelled Met-tRNAfMet and exogenous RNA templates, and assaying the formation of labelled 80S initiation complexes in the presence of GTP, or labelled 40S initiation complexes in the presence of a non-hydrolysable analogue of GTP. Initiation complex formation on globin mRNA, or on capped viral RNAs such as papaya mosaic virus RNA and tobacco mosaic virus RNA, was strongly stimulated by ATP. In contrast, initiation complex formation on (uncapped) encephalomyocarditis virus RNA was uninfluenced by the presence or absence of ATP, which may be correlated with the recent evidence for scanning-independent internal initiation on this viral RNA. In addition, initiation complex formation on uncapped cowpea mosaic virus RNA and on poly(A,U,G) was only slightly stimulated by ATP, much less than in the case of the capped RNAs. These results suggest that most of the ATP hydrolysed during translation initiation is consumed in cap-dependent processes, probably in unwinding the mRNA, and relatively little in the actual migration or scanning of 40S subunits along the mRNA.
利用经凝胶过滤的兔网织红细胞裂解物，将其与标记的甲硫氨酰 - tRNAfMet和外源RNA模板一起孵育，并在存在GTP的情况下检测标记的80S起始复合物的形成，或在存在GTP的不可水解类似物的情况下检测标记的40S起始复合物的形成，以此来测试真核生物mRNA翻译起始对ATP的需求。ATP强烈刺激了珠蛋白mRNA上，或有帽病毒RNA（如番木瓜花叶病毒RNA和烟草花叶病毒RNA）上起始复合物的形成。相比之下，（无帽的）脑心肌炎病毒RNA上起始复合物的形成不受ATP存在与否的影响，这可能与最近关于该病毒RNA进行不依赖扫描的内部起始的证据相关。此外，无帽的豇豆花叶病毒RNA和聚（A，U，G）上起始复合物的形成仅受到ATP的轻微刺激，远低于有帽RNA的情况。这些结果表明，翻译起始过程中水解的大部分ATP消耗于依赖帽的过程，可能用于解开mRNA，而40S亚基沿mRNA实际迁移或扫描所消耗的ATP相对较少。
