Ray Sibnath, Yumak Hasan, Domashevskiy Artem, Khan Mateen A, Gallie Daniel R, Goss Dixie J
Department of Chemistry, Hunter College and the Graduate Center, City University of New York, New York, New York 10021, USA.
J Biol Chem. 2006 Nov 24;281(47):35826-34. doi: 10.1074/jbc.M605762200. Epub 2006 Sep 29.
The 5'-leader of tobacco etch virus (TEV) genomic RNA directs the efficient translation from the naturally uncapped viral RNA. The TEV 143-nt 5'-leader folds into a structure that contains two domains, each of which contains RNA pseudoknots. The 5'-proximal pseudoknot 1 (PK1) is necessary to promote cap-independent translation (Zeenko, V., and Gallie, D. R. (2005) J. Biol. Chem. 280, 26813-26824). During the translation initiation of cellular mRNAs, eIF4G functions as an adapter that recruits many of the factors involved in stimulating 40 S ribosomal subunit binding to an mRNA. Two related but highly distinct eIF4G proteins are expressed in plants, animals, and yeast. The two plant eIF4G isoforms, referred to as eIF4G and eIFiso4G, differ in size (165 and 86 kDa, respectively) and their functional differences are still unclear. Although eIF4G is required for the translation of TEV mRNA, it is not known if eIF4G binds directly to the TEV RNA itself or if other factors are required. To determine whether binding affinity and isoform preference correlates with translational efficiency, fluorescence spectroscopy was used to measure the binding of eIF4G, eIFiso4G, and their complexes (eIF4F and eIFiso4F, respectively) to the TEV 143-nt 5'-leader (TEV1-143) and a shorter RNA that contained PK1. A mutant (i.e. S1-3) in which the stem of PK1 was disrupted resulting in impaired cap-independent translation, was also tested. These studies demonstrate that eIF4G binds TEV1-143 and PK1 RNA with approximately 22-30-fold stronger affinity than eIFiso4G. eIF4G and eIF4F bind TEV1-143 with similar affinity, whereas eIFiso4F binds with approximately 6-fold higher affinity than eIFiso4G. The binding affinity of eIF4G, eIF4F, and eIFiso4G to S1-3 was reduced by 3-5-fold, consistent with the reduction in the ability of this mutant to promote cap-independent translation. Temperature-dependent binding studies revealed that binding of the TEV 5'-leader to these initiation factors has a large entropic contribution. Overall, these results demonstrate the first direct interaction of eIF4G with the TEV 5'-leader in the absence of other initiation factors. These data correlate well with the observed translational data and provide more detailed information on the translational strategy of potyviruses.
烟草蚀纹病毒（TEV）基因组RNA的5′端前导序列指导天然无帽病毒RNA的高效翻译。TEV的143个核苷酸的5′端前导序列折叠成一种结构，该结构包含两个结构域，每个结构域都含有RNA假结。5′近端假结1（PK1）对于促进不依赖帽的翻译是必需的（泽恩科，V.，和加利，D.R.（2005）《生物化学杂志》280，26813 - 26824）。在细胞mRNA的翻译起始过程中，eIF4G作为一种衔接蛋白发挥作用，招募许多参与刺激40S核糖体亚基与mRNA结合的因子。在植物、动物和酵母中表达两种相关但高度不同的eIF4G蛋白。两种植物eIF4G同工型，分别称为eIF4G和eIFiso4G，大小不同（分别为165和86 kDa），它们的功能差异尚不清楚。虽然eIF4G是TEV mRNA翻译所必需的，但尚不清楚eIF4G是否直接与TEV RNA本身结合，或者是否需要其他因子。为了确定结合亲和力和同工型偏好是否与翻译效率相关，采用荧光光谱法测量eIF4G、eIFiso4G及其复合物（分别为eIF4F和eIFiso4F）与TEV的143个核苷酸的5′端前导序列（TEV1 - 143）以及包含PK1的较短RNA的结合。还测试了一个突变体（即S1 - 3），其中PK1的茎被破坏，导致不依赖帽的翻译受损。这些研究表明，eIF4G与TEV1 - 143和PK1 RNA的结合亲和力比eIFiso4G强约22 - 30倍。eIF4G和eIF4F以相似的亲和力结合TEV1 - 143，而eIFiso4F的结合亲和力比eIFiso4G高约6倍。eIF4G、eIF4F和eIFiso4G与S1 - 3的结合亲和力降低了3 - 5倍，这与该突变体促进不依赖帽翻译的能力降低一致。温度依赖性结合研究表明，TEV 5′端前导序列与这些起始因子的结合有很大的熵贡献。总体而言，这些结果证明了在没有其他起始因子的情况下，eIF4G与TEV 5′端前导序列的首次直接相互作用。这些数据与观察到的翻译数据很好地相关，并提供了关于马铃薯Y病毒翻译策略的更详细信息。
