Kolupaeva V G, Hellen C U, Shatsky I N
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
RNA. 1996 Dec;2(12):1199-212.
Initiation of translation of a subset of eukaryotic mRNAs results from internal ribosomal entry. This process is exemplified by encephalomyocarditis virus (EMCV), which contains an internal ribosomal entry site (IRES) within its 5' nontranslated region that is approximately 450-nt long and consists of a series of stem-loops designated H-L. We have previously identified a cellular 58-kDa polypeptide that binds specifically to this IRES and that is implicated in its function as the pyrimidine tract-binding protein PTB. We have now mapped PTB binding sites directly on the IRES elements of EMCV and the related foot-and-mouth disease virus (FMDV) using structure-specific enzymatic probes and base-specific chemical probes. PTB bound to six sites on the EMCV IRES: site 1 (UCUU401) is upstream of domain H, site 2 is the basal helix of domain H (nt 407-410 and 440-443), site 3 (UCUUU423) is the apical loop of domain H, site 4 is the apical helix and adjacent internal bulged loop of domain K, site 5 (CUUUA750) is the apical loop of domain K, and site 6 (CCUUU815) is downstream of domain L. PTB bound to sites on the FMDV IRES that correspond precisely to EMCV sites 3, 5, and 6. These sites have the consensus sequence CUUU and form two groups that are located near to the 5' and 3' borders of these IRES elements. Their position, and the effects of mutation of them on IRES function are consistent with PTB's role in IRES-mediated initiation being to bind to multiple sites in the IRES, thereby stabilizing a specific active conformation.
真核生物部分mRNA的翻译起始是由核糖体内部进入引发的。脑心肌炎病毒（EMCV）就是这一过程的典型例子，其5'非翻译区包含一个约450个核苷酸长的核糖体内部进入位点（IRES），该位点由一系列命名为H-L的茎环组成。我们之前鉴定出一种细胞58 kDa多肽，它能特异性结合该IRES，并作为嘧啶序列结合蛋白PTB参与其功能。现在，我们使用结构特异性酶探针和碱基特异性化学探针，将PTB结合位点直接定位在EMCV和相关口蹄疫病毒（FMDV）的IRES元件上。PTB与EMCV IRES上的六个位点结合：位点1（UCUU401）在结构域H的上游，位点2是结构域H的基部螺旋（核苷酸407 - 410和440 - 443），位点3（UCUUU423）是结构域H的顶端环，位点4是结构域K的顶端螺旋和相邻的内部凸起环，位点5（CUUUA750）是结构域K的顶端环，位点6（CCUUU815）在结构域L的下游。PTB与FMDV IRES上的位点结合，这些位点与EMCV的位点3、5和6精确对应。这些位点具有共有序列CUUU，并形成两组，位于这些IRES元件的5'和3'边界附近。它们的位置以及对它们的突变对IRES功能的影响与PTB在IRES介导的起始过程中的作用一致，即结合到IRES中的多个位点，从而稳定特定的活性构象。
