Grigoriadou Christina, Marzi Stefano, Pan Dongli, Gualerzi Claudio O, Cooperman Barry S
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.
J Mol Biol. 2007 Oct 26;373(3):551-61. doi: 10.1016/j.jmb.2007.07.031. Epub 2007 Aug 2.
IF3 has a fidelity function in the initiation of translation, inducing the dissociation of fMet-tRNA(fMet) from the 30 S initiation complexes (30SIC) containing a non-canonical initiation triplet (e.g. AUU) in place of a canonical initiation triplet (e.g., AUG). IF2 has a complementary role, selectively promoting initiator tRNA binding to the ribosome. Here, we used parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light-scattering, and changes in intensities of fluorophore-labeled IF2 and fMet-tRNA(fMet) to determine the effects on both 30SIC formation and 30SIC conversion to 70 S initiation complexes (70SIC) of (a) substituting AUG with AUU, and/or (b) omitting IF3, and/or (c) replacing GTP with the non-hydrolyzable analog GDPCP. We demonstrate that the presence or absence of IF3 has, at most, minor effects on the rate of 30SIC formation using either AUG or AUU as the initiation codon, and conclude that the high affinity of IF2 for both 30 S subunit and initiator tRNA overrides any perturbation of the codon-anticodon interaction resulting from AUU for AUG substitution. In contrast, replacement of AUG by AUU leads to a dramatic reduction in the rate of 70SIC formation from 30SIC upon addition of 50 S subunits. Interpreting our results in the framework of a quantitative kinetic scheme leads to the conclusion that, within the overall process of 70SIC formation, the step most affected by substituting AUU for AUG involves the conversion of an initially labile 70 S ribosome into a more stable complex. In the absence of IF3, the difference between AUG and AUU largely disappears, with each initiation codon affording rapid 70SIC formation, leading to the hypothesis that it is the rate of IF3 dissociation from the 70 S ribosome during IC70S formation that is critical to its fidelity function.
IF3在翻译起始过程中具有保真功能，可诱导甲酰甲硫氨酰 - tRNA（fMet - tRNA(fMet)）从含有非经典起始三联体（如AUU）而非经典起始三联体（如AUG）的30S起始复合物（30SIC）上解离。IF2具有互补作用，可选择性地促进起始tRNA与核糖体结合。在此，我们通过对GTP水解、Pi释放、光散射以及荧光团标记的IF2和fMet - tRNA(fMet)强度变化进行平行快速动力学测量，以确定以下因素对30SIC形成以及30SIC转化为70S起始复合物（70SIC）的影响：(a) 用AUU替代AUG，和/或(b) 省略IF3，和/或(c) 用不可水解的类似物GDPCP替代GTP。我们证明，无论使用AUG还是AUU作为起始密码子，IF3的存在与否对30SIC形成速率的影响至多较小，并得出结论，即IF2对30S亚基和起始tRNA的高亲和力克服了因用AUU替代AUG而导致的密码子 - 反密码子相互作用的任何扰动。相比之下，用AUU替代AUG会导致在添加50S亚基后从30SIC形成70SIC的速率显著降低。在定量动力学方案的框架内解释我们的结果得出结论，在70SIC形成的整个过程中，用AUU替代AUG影响最大的步骤涉及将最初不稳定的70S核糖体转化为更稳定的复合物。在没有IF3的情况下，AUG和AUU之间的差异基本消失，每个起始密码子都能实现快速的70SIC形成，从而提出这样的假设，即在IC70S形成过程中IF3从70S核糖体上解离的速率对其保真功能至关重要。
