Ribosomal scanning and initiation codon selection

描述（由申请人提供）：核糖体扫描模型假定起始密码子选择的三步机制。包含40 S亚基、起始tRNA和起始因子（eIF）eIF 2、3、1和1A的43 S预起始复合物首先结合mRNA的5 '端，然后沿5'非翻译区（5 '-UTR）向下游沿着扫描，直到它们在有利的环境中遇到第一个AUG三联体。扫描机制需要持续保护以防止5 '-UTR中的三联体与起始tRNA的反密码子之间的部分碱基配对。在维持起始密码子选择的保真度中的关键作用属于eIFl，其使得43 S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<8 nt的AUG三联体。从mRNA的5 '端开始。elF 1在40 S亚基上的位置表明elF 1通过诱导起始复合物中的构象变化间接地执行其功能。为了研究eIFl的作用机制，我们将（i）通过确定eIFl对mRNA在40 S亚基上的位置的影响来研究起始复合物中的构象变化，（ii）分析eIFl解离异常核糖体复合物的机制，和（iii）研究eIFl参与起始tRNA的选择。为了深入了解elF 1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IF 3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）和生物化学（UV交联，足迹和羟基自由基裂解）技术的强大组合，以获得一个明确的建筑模型的43/48 S核糖体复合物。该模型将建立起始因子、起始tRNA和mRNA在40 S亚基上的相对位置，并将揭示mRNA穿线通道的结构。

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.