SCANNING PROCESS IN EUKARYOTIC TRANSLATION

翻译起始需要真核起始因子（eIF）2和3将起始子tRNA结合到40 S核糖体亚基上，并需要eIF 4A、4 B和4F将产生的43 S复合物连接到mRNA的5'末端。 在从纯化的翻译组分体外重建的反应中，该复合物在没有eIFs 1和1A的情况下不能到达β-珠蛋白起始密码子。 该建议的目的是确定43 S复合物如何从5'端扫描到起始密码子，以及它如何与60 S亚基连接形成活性80 S核糖体。我们将使用突变的mRNA来验证这些反应的起始符合扫描和起始密码子识别的实验标准。 如果需要额外的活性，将结合适当的功能测定从允许细胞提取物中纯化必要的因子。 相同的策略将用于纯化和鉴定亚基连接所需的因子。 然后，使用荧光共振能量转移直接测定核糖体扫描，以确定标记的43 S复合物与连接在模型mRNA上定义位置的荧光团的接近度。 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。 在完全非结构化的5 'NTR上起始期间的因子省略将用于区分核糖体和相关eIF 4A扫描期间的ATP水解。 eIFs 1和1A与其他翻译组分（mRNA，40 S亚基和eIF 3）之间的相互作用的结构基础的特点将阐明这些因素在扫描，起始密码子识别和解离异常核糖体复合物从mRNA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.