Kinetic and thermodynamic analysis of the role of start codon/anticodon base pairing during eukaryotic translation initiation
Kinetic and thermodynamic analysis of the role of start codon/anticodon base pairing during eukaryotic translation initiation
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DOI：
10.1261/rna.1318509
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发表时间：
2009-01-01
期刊：
影响因子：
4.5
通讯作者：
Lorsch, Jon R.
中科院分区：
文献类型：
--
作者：
Kolitz, Sarah E.;Takacs, Julie E.;Lorsch, Jon R.
起始密码子识别是蛋白质合成起始的关键事件。为了深入了解真核生物中起始密码子识别的机制，我们使用酵母重组起始系统分离Met-tRNA（i）中心点eIF 2中心点GTP三元复合物（TC）与40 S亚基结合的步骤。我们研究了在mRNA起始密码子和起始甲硫氨酰tRNA反密码子中存在碱基变化的情况下这一步骤的动力学和热力学，以研究碱基配对和序列对所得43 S中心点mRNA复合物稳定性的影响。我们观察到，三个碱基对的形成，而不是它们的身份，是TC结合稳定性的关键决定因素，这表明该步骤的序列AUG没有什么内在的特殊性。令人惊讶的是，TC与40 S亚基结合的速率常数具有强烈的密码子依赖性，而TC从43 S中心点mRNA复合物解离的速率常数则不具有密码子依赖性。这些数据表明，一个模型中，在最初的扩散有限遇到TC与40 S亚基，形成三个匹配的起始密码子/反密码子碱基对触发的构象变化，锁定复杂的稳定状态。这一诱导契合机制支持了43 S复合物对起始密码子的识别诱导了从开放状态到封闭状态的构象变化，从而阻止了mRNA的沿着运动。
Start codon recognition is a crucial event in the initiation of protein synthesis. To gain insight into the mechanism of start codon recognition in eukaryotes, we used a yeast reconstituted initiation system to isolate the step of Met-tRNA(i)center dot eIF2 center dot GTP ternary complex (TC) binding to the 40S subunit. We examined the kinetics and thermodynamics of this step in the presence of base changes in the mRNA start codon and initiator methionyl tRNA anticodon, in order to investigate the effects of base pairing and sequence on the stability of the resulting 43S center dot mRNA complex. We observed that the formation of three base pairs, rather than their identities, was the key determinant of stability of TC binding, indicating that nothing is inherently special about the sequence AUG for this step. Surprisingly, the rate constant for TC binding to the 40S subunit was strongly codon dependent, whereas the rate constant for TC dissociation from the 43S center dot mRNA complex was not. The data suggest a model in which, after the initial diffusion-limited encounter of TC with the 40S subunit, the formation of three matching start codon/anticodon base pairs triggers a conformational change that locks the complex into a stable state. This induced-fit mechanism supports the proposal that initiation codon recognition by the 43S complex induces a conformational change from an open state to a closed one that arrests movement along the mRNA.