The Roles of eIF4G and Associated Factors in Ribosomal Attachment and Scanning During Translation Initiation

真核细胞起始因子(EIF)4G通过与这两个步骤所需的因子结合来协调核糖体结合以及随后在mRNA的5‘NTR上进行核糖体扫描。EIF4G是eIF4F的亚基，eIF4F还含有eIF4E(帽结合蛋白)和eIF4A(解旋酶)亚基。本项目将表征eIF4G及相关因子在使用mRNA的这两个启动阶段中的作用，这两个阶段取决于核糖体结合的5‘末端帽或内部核糖体进入位点(IRES)。将40S核糖体亚基装载到mRNA上可能涉及通过结合eIF4F局部解离mRNA以及eIF4G与核糖体相关的eIF3之间的相互作用。EIF4G与RNA、eIF4A和eIF3的相互作用在核糖体附着和扫描中的作用将通过将生化分析(如eIF4G对eIF4A解旋酶活性的影响)与模型mRNAs的起始重构相结合来表征。Ded1p，另一种与扫描有关的RNA解旋酶的作用也将被描述。脑心肌炎病毒(EMCV)IRES的启动需要eIF4G/4A的特异性结合。EIF4G/4A识别这一IRES的机制、特定IRES反式作用因子增强eIF4G/4A与相关IRESS的结合以及eIF4G/4A/IRES复合体促进核糖体附着的机制将被确定。EIF4F的帽结合活性在有丝分裂和凋亡过程中被下调，当一些细胞内含有IRES的mRNAs保持活性时。当eIF4E无效时，这些IRESS类似于EMCV与eIF4G/4A相互作用以促进核糖体结合的假设将得到验证。这些研究将为特定mRNAs的选择性翻译机制提供洞察力。

Eukaryotic initiation factor (eIF) 4G coordinates ribosomal binding and subsequent ribosomalscanning on the 5'NTR of an mRNA by binding to factors that are required for both steps. eIF4G is asubunit of eIF4F, which also has eIF4E (cap-binding protein) and eIF4A (helicase) subunits. This projectwill characterize the roles of eIF4G and associated factors in these two stages of initiation using mRNAsthat depend on either the 5'-terminal cap or an internal ribosomal entry site (IRES) for ribosomalbinding. Loading of the 40S ribosomal subunit onto mRNA may involve local unwinding of mRNA bybound eIF4F and interaction between eIF4G and ribosome-associated eIF3. The roles of eIF4G'sinteractions with RNA, eIF4A and eIF3 in ribosomal attachment and scanning will be characterized byintegrating biochemical assays (such as eIF4G's influence on eIF4A's helicase activity) withreconstitution of initiation on model mRNAs. The role of Ded1p, another RNA helicase that has beenimplicated in scanning will also be characterized. Initiation on the encephalomyocarditis virus (EMCV)IRES requires specific binding by eIF4G/4A. The mechanisms of recognition of this IRES by eIF4G/4A, ofenhancement of binding of eIF4G/4A to related IRESs by specific IRES trans-acting factors, and ofpromotion of ribosomal attachment by the eIF4G/4A/IRES complex will be determined. The cap-bindingactivity of eIF4F is down-regulated during mitosis and apoptosis when some cellular IRES-containingmRNAs remain active. The hypothesis that these IRESs resemble EMCV in interacting with eIF4G/4A topromote ribosomal binding when eIF4E is inactive will be tested. These studies will provide insightsinto mechanisms for selective translation of specific mRNAs.