Kolupaeva V G, Pestova T V, Hellen C U
Department of Microbiology and Immunology, State University of New York Health Science Center at Brooklyn, 11203, USA.
RNA. 2000 Dec;6(12):1791-807. doi: 10.1017/s1355838200000662.
Most eukaryotic mRNAs require the cap-binding complex elF4F for efficient initiation of translation, which occurs as a result of ribosomal scanning from the capped 5' end of the mRNA to the initiation codon. A few cellular and viral mRNAs are translated by a cap and end-independent mechanism known as internal ribosomal entry. The internal ribosome entry site (IRES) of classical swine fever virus (CSFV) is approximately 330 nt long, highly structured, and mediates internal initiation of translation with no requirement for elF4F by recruiting a ribosomal 43S preinitiation complex directly to the initiation codon. The key interaction in this process is the direct binding of ribosomal 40S subunits to the IRES to form a stable binary complex in which the initiation codon is positioned precisely in the ribosomal P site. Here, we report the results of analyses done using enzymatic footprinting and mutagenesis of the IRES to identify structural components in it responsible for precise binding of the ribosome. Residues flanking the initiation codon and extending from nt 363-391, a distance equivalent to the length of the 40S subunit mRNA-binding cleft, were strongly protected from RNase cleavage, as were nucleotides in the adjacent pseudoknot and in the more distal subdomain IIId1. Ribosomal binding and IRES-mediated initiation were abrogated by disruption of helix 1b of the pseudoknot and very severely reduced by mutation of the protected residues in IIId1 and by disruption of domain IIIa. These observations are consistent with a model for IRES function in which binding of the region flanking the initiation codon to the decoding region of the ribosome is determined by multiple additional interactions between the 40S subunit and the IRES.
大多数真核生物mRNA需要帽结合复合物elF4F来高效启动翻译，翻译过程是核糖体从mRNA的5'端帽结构扫描至起始密码子的结果。一些细胞和病毒mRNA通过一种不依赖帽和末端的机制进行翻译，即内部核糖体进入。经典猪瘟病毒（CSFV）的内部核糖体进入位点（IRES）约330个核苷酸长，结构高度复杂，通过直接将核糖体43S起始前复合物募集到起始密码子，介导不依赖elF4F的内部翻译起始。此过程中的关键相互作用是核糖体40S亚基直接与IRES结合，形成稳定的二元复合物，其中起始密码子精确位于核糖体P位点。在此，我们报告了使用酶足迹法和IRES诱变进行分析的结果，以确定其中负责核糖体精确结合的结构成分。起始密码子两侧以及从核苷酸363 - 391延伸的残基（相当于40S亚基mRNA结合裂隙的长度）受到强烈保护，免受核糖核酸酶切割，相邻假结和更远端的亚结构域IIId1中的核苷酸也是如此。假结的螺旋1b被破坏会消除核糖体结合和IRES介导的起始，IIId1中受保护残基的突变以及结构域IIIa的破坏会使其严重减少。这些观察结果与IRES功能模型一致，即起始密码子侧翼区域与核糖体解码区域的结合由40S亚基和IRES之间的多个额外相互作用决定。
