Ribosomal scanning and initiation codon selection

描述（由申请人提供）：核糖体扫描模型假设起始密码子选择的三步机制。43S预启动复合物包括40S亚基、启动物tRNA和启动因子（elF） elF 2、3、1和1A首先结合到mRNA的5‘端，然后沿着5’-非翻译区（5'-UTR）向下扫描，直到它们在有利的环境中遇到第一个AUG三元组。扫描机制需要持续保护，防止5'-UTR三联体与启动器tRNA的反密码子之间的部分碱基修复。elF1在维持起始密码子选择的保真度中起着关键作用，它使43S复合物能够拒绝密码子-反密码子错配，识别起始密码子的上下文，并区分距离mRNA 5'端<8 nt的AUG三联体。elF1在40S亚基上的位置表明，elF1通过诱导起始复合物的构象变化间接发挥作用。为了研究eIFl的作用机制，我们将(i)通过确定elF1对40S亚基mRNA位置的影响来研究起始复合物的构象变化，（ii）分析elF1解离异常核糖体复合物的机制，（iii）研究elFl参与启动物tRNA的选择。为了深入了解elF1的进化起源，我们将分别确定其原核结构和功能同源物YciH和起始因子IF3在原核和真核翻译起始中的活性。为了提供理解核糖体扫描和起始密码子识别的分子机制的基础，我们将应用生物物理（低温电子显微镜，热氚轰击）和生化（紫外交联，脚印和羟基自由基切割）技术的强大组合来获得一个定义良好的43/48S核糖体复合物的结构模型。由此建立的模型将建立起始因子、引发剂tRNA和mRNA在40S亚基上的相对位置，并揭示mRNA线程通道的结构。

DESCRIPTION (provided by applicant): The ribosomal scanning model postulates a three-step mechanism for initiation codon selection. 43S preinitiation complexes comprising 40S subunits, initiator tRNA and initiation factors (elF) elFs 2, 3, 1 and 1A first bind to the 5'-end of mRNA and then scan downstream along the 5'-untranslated region (5'-UTR) until they encounter the first AUG triplet in a favorable context. The scanning mechanism requires continuous protection against partial basepairing between triplets in the 5'-UTR and the anticodon of initiator tRNA. A key role in maintaining the fidelity of initiation codon selection belongs to elF1, which enables 43S complexes to reject codon-anticodon mismatches, to recognize the context of the initiation codon and to discriminate against AUG triplets that are <8 nt. from the 5'-end of mRNA. The position of elF1 on the 40S subunit suggests that elF1 performs its function indirectly by inducing conformational changes in the initiation complex. To investigate the mechanism of eIFl's action we shall (i) study conformational changes in initiation complexes by determining the influence of elF1 on the position of mRNA on the 40S subunit, (ii) analyze the mechanism of dissociation of aberrant ribosomal complexes by elF1, and (iii) investigate elFl's involvement in selection of initiator tRNA. To get insights into evolutionary origin of elF1 we shall determine the activities of its prokaryotic structural and functional homologues, YciH and initiation factor IF3, respectively, in both prokaryotic and eukaryotic translation initiation. To provide the basis for understanding the molecular mechanism of ribosomal scanning and initiation codon recognition we shall apply a powerful combination of biophysical (cryo-electron microscopy, hot tritium bombardment) and biochemical (UV cross-linking, foot-printing and hydroxyl radical cleavage) techniques to obtain a well-defined architectural model of 43/48S ribosomal complexes. The resulting model will establish the relative positions of initiation factors, initiator tRNA and mRNA on the 40S subunit, and will reveal the structure of mRNA threading channel.