Borovjagin A V, Ezrokhi M V, Rostapshov V M, Bystrova T F, Shatsky I N
A.N. Belozersky Laboratory, Moscow State University, USSR.
Nucleic Acids Res. 1991 Sep 25;19(18):4999-5005. doi: 10.1093/nar/19.18.4999.
Various derivatives of the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) RNA have been used to analyze by UV-cross-linking its interaction with mRNA binding proteins from ascites carcinoma Krebs-2 cells. A doublet of proteins with Mr 58 and 60 kD bound to two regions of the IRES. One site is centered at nt 420-421 of EMCV RNA whereas the other is located between nt 315-377. Both sites form hairpin structures, the loops of which contain UCUUU motif, conserved among cardio- and aphthoviruses. The interaction of p58 and p60 with IRES is affected by the integrity of the stem-loop structure proximal to the start AUG codon (nts 680-787), although, under similar conditions, cross-linking of these proteins to this region was not detected. Deletions in the main recognition site of p58 strongly reduce the initiation activity of the IRES in vitro. However, elimination of p58 (p60) binding by these mutations does not completely abolish the ability of the IRES to direct polypeptide synthesis starting from the authentic AUG codon. The IRES can be assembled in vitro from two covalently unlinked transcripts, one containing the target site for p58 and the other encompassing the remaining part of the IRES fused to a reporter gene, resulting in considerable restoration of its activity. Implications of these findings for the mechanism of initiation resulting from internal entry of ribosomes are discussed.
脑心肌炎病毒（EMCV）RNA的内部核糖体进入位点（IRES）的各种衍生物已被用于通过紫外线交联分析其与腹水癌Krebs-2细胞中mRNA结合蛋白的相互作用。有两个Mr为58和60 kD的蛋白质与IRES的两个区域结合。一个位点位于EMCV RNA的nt 420 - 421处，而另一个位于nt 315 - 377之间。这两个位点均形成发夹结构，其环中含有UCUUU基序，在心脏病毒和口蹄疫病毒中保守。p58和p60与IRES的相互作用受起始AUG密码子近端（nts 680 - 787）茎环结构完整性的影响，尽管在相似条件下未检测到这些蛋白质与该区域的交联。p58主要识别位点的缺失在体外强烈降低IRES的起始活性。然而，通过这些突变消除p58（p60）结合并不能完全消除IRES从真实AUG密码子起始多肽合成的能力。IRES可以在体外由两个共价不相连的转录本组装而成，一个包含p58的靶位点，另一个包含与报告基因融合的IRES其余部分，从而使其活性得到显著恢复。讨论了这些发现对核糖体内部进入导致的起始机制的意义。
