SCANNING PROCESS IN EUKARYOTIC TRANSLATION

翻译启动需要真核细胞起始因子(EIFS)2和3将启动子tRNA与40S核糖体亚基结合，而EIFS4A、4B和4F则需要将得到的43S复合体结合到mRNA的5‘端。在由纯化的翻译成分在体外重组的反应中，该复合体没有达到没有EIFS1和1A的β-珠蛋白起始密码子。这个提议的目的是确定43S复合体如何从5‘端扫描到起始密码子，以及它如何与60S亚基连接形成活性的80S核糖体。我们将使用突变的mRNAs来验证这些反应中的启动是否符合扫描和启动密码子识别的实验标准。如果需要额外的活性，将从允许的细胞提取液中提纯必要的因子，并结合适当的功能分析。同样的策略将被用来纯化和确定亚基连接所需的因素。然后，核糖体扫描将直接使用荧光共振能量转移进行分析，以确定标记的43S复合体与固定在模型mRNA上特定位置的荧光团的接近程度。这将使我们能够确定扫描是否涉及核糖体对5‘ntR的逐个碱基检查。在完全非结构的5‘NTR启动过程中的因子遗漏将被用来区分核糖体扫描期间的ATP水解和相关的eIF4A。EIFS1和1A与其他翻译成分(mRNA、40S亚基和eIF3)相互作用的结构基础将被描述，以阐明这些因素在扫描、起始密码子识别和从mRNA中解离异常核糖体复合体中的重要作用。

Translation initiation requires eukaryotic initiation factors (eIFs) 2 and 3 to bind initiator tRNA to a 40S ribosomal subunit and eIFs 4A, 4B and 4F to attach the resulting 43S complex to the 5' end of an mRNA. In a reaction reconstituted in vitro from purified translation components, this complex did not reach the beta-globin initiation codon without eIFs 1 and 1A. The aim of this proposal is to determine how the 43S complex scans from the 5' end to the initiation codon and how it is then joined to a 60S subunit to form an active 80S ribosome. We shall use mutant mRNAs to verify that initiation in these reactions meets experimental criteria for scanning and initiation codon recognition. If additional activities are required, necessary factors will be purified from permissive cell extracts in conjunction with appropriate assays of function. The same strategy will be used to purify and identify factors required for subunit joining. Ribosomal scanning will then be assayed directly using fluorescence resonance energy transfer to determine the proximity of labelled 43S complexes to fluorophores attached at defined positions on model mRNAs. This will enable us to determine whether scanning involves base-by-base inspection of a 5'NTR by the ribosome. Factor omission during initiation on a wholly unstructured 5'NTR will be used to differentiate between ATP hydrolysis during scanning by ribosomes and by associated eIF4A. The structural basis of interactions between eIFs 1 and 1A with other translation components (mRNA, 40S subunits and eIF3) will be characterized to elucidate the essential roles of these factors in scanning, initiation codon recognition and dissociating aberrant ribosomal complexes from mRNA.