Konarska M, Filipowicz W, Domdey H, Gross H J
Eur J Biochem. 1981 Feb;114(2):221-7. doi: 10.1111/j.1432-1033.1981.tb05139.x.
The sequence of the 5'-terminal leader fragment preceding the AUG codon in the RNA of tobacco mosaic virus (TMV), tomato strain, SPS isolate, has been determined. This RNA, similarly to the RNAs of the U1 and Dahlemense strains of TMV [Kukla et al. (1979) Eur. J. Biochem. 98, 61--66] has the 7-methylguanosine(5')triphospho(5')guanosine cap separated from the initiation codon by a long stretch of nucleotides devoid of guanosine residues. The RNase-T1-resistant 73-nucleotide-long leader fragment of TMV RNA from the SPS isolate was assayed for its ability to interact with eukaryotic and prokaryotic ribosomes. The linear fragment, labelled either at its 5' or 3' end, efficiently formed disome initiation complexes when incubated with wheat-germ protein-synthesis extract. In contrast to its linear counterpart, the circular covalently closed RNA leader fragment, obtained in a reaction catalysed by T4 RNA ligase, was unable to interact with wheat germ ribosomes. Both kinds of leader fragment bound equally well to Escherichia coli 70-S ribosomes. The results offer further support to the notion that in eukaryotic initiation the free 5' end (either capped or uncapped) is required for mRNA interaction with ribosomes. Furthermore, they suggest that both ribosomes found in disome initiation complexes with the TMV RNA leader fragment enter the mRNA sequentially via the free 5' terminus.
已确定烟草花叶病毒（TMV）番茄株系SPS分离物RNA中AUG密码子之前5'-末端前导片段的序列。与TMV的U1和达勒姆森株系的RNA [库克拉等人（1979年），欧洲生物化学杂志98，61 - 66] 相似，该RNA具有7-甲基鸟苷（5'）三磷酸（5'）鸟苷帽，其与起始密码子之间被一段缺乏鸟苷残基的长核苷酸序列隔开。对来自SPS分离物的TMV RNA的73个核苷酸长的、抗核糖核酸酶T1的前导片段与真核和原核核糖体相互作用的能力进行了测定。当与小麦胚芽蛋白质合成提取物一起孵育时，在其5'或3'末端标记的线性片段有效地形成了二体起始复合物。与线性对应物相反，在T4 RNA连接酶催化的反应中获得的环状共价闭合RNA前导片段不能与小麦胚芽核糖体相互作用。两种前导片段与大肠杆菌70-S核糖体的结合效果相同。这些结果进一步支持了以下观点：在真核起始过程中，mRNA与核糖体相互作用需要游离的5'末端（无论有无帽结构）。此外，它们表明，与TMV RNA前导片段形成二体起始复合物的两个核糖体都是通过游离的5'末端依次进入mRNA的。
