Functional characterization of the role of the N-terminal domain of the c/Nip1 subunit of eukaryotic initiation factor 3 (eIF3) in AUG recognition.
Functional characterization of the role of the N-terminal domain of the c/Nip1 subunit of eukaryotic initiation factor 3 (eIF3) in AUG recognition.
复制标题
DOI：
10.1074/jbc.m112.386656
复制
发表时间：
2012-08-17
期刊：
影响因子：
--
通讯作者：
Valášek L
中科院分区：
文献类型：
--
作者：
Karásková M;Gunišová S;Herrmannová A;Wagner S;Munzarová V;Valášek L
背景：AUG识别受多种启动因子(EIF)的促进。结果：eIF5与eIF3c/Nip1的N端相互作用，促进预启动复合体的组装，eIF1结合紧随其后的区域。结论：eIF1与c/Nip1的结合对其40 S核糖体募集和AUG选择同样重要。意义：理解起始密码子选择是基因表达研究中的关键，它设定了解码的阅读框架。在真核生物中，对于要合成的蛋白质，40 S亚基必须首先扫描mRNA5‘端非编码区，直到它遇到AUG起始密码子。先前已经确定了几个确保AUG识别高保真的启动因素，包括eIF1A、eIF1、eIF2和eIF5。此外，还建议eIF3协调它们在这一过程中的功能，并促进它们与40个S亚基的初始结合。在这里，我们对eIF3c/Nip1亚基中介导eIF3与eIF1和eIF5结合的N-末端结构域(NTD)的几个片段进行了半随机突变，以研究eIF3参与这些反应的分子机制。分离出了三种主要的突变替换或内部缺失，它们要么影响预起始复合体(PIC)的组装，要么扫描AUG，或者两者兼而有之。我们发现eIF5与极端的c/Nip1-NTD(残基1-45)结合，破坏这种相互作用主要影响PIC的形成。EIF1与紧随其后的区域(60-137)相互作用，改变这种联系将解除对AUG的识别。综上所述，我们的数据表明，eIF1与c/Nip1-NTD的结合对于它最初被招募到PICS和选择翻译起始位置的正确功能同样重要。
Background: AUG recognition is promoted by several initiation factors (eIFs). Results: eIF5 interacts with the extreme N terminus of eIF3c/Nip1 to promote pre-initiation complex assembly, and eIF1 binds the region that immediately follows. Conclusion: eIF1 binding to c/Nip1 is equally important for its 40 S ribosome recruitment and AUG selection. Significance: Understanding start codon selection that sets the reading frame for decoding is key in gene expression studies. In eukaryotes, for a protein to be synthesized, the 40 S subunit has to first scan the 5′-UTR of the mRNA until it has encountered the AUG start codon. Several initiation factors that ensure high fidelity of AUG recognition were identified previously, including eIF1A, eIF1, eIF2, and eIF5. In addition, eIF3 was proposed to coordinate their functions in this process as well as to promote their initial binding to 40 S subunits. Here we subjected several previously identified segments of the N-terminal domain (NTD) of the eIF3c/Nip1 subunit, which mediates eIF3 binding to eIF1 and eIF5, to semirandom mutagenesis to investigate the molecular mechanism of eIF3 involvement in these reactions. Three major classes of mutant substitutions or internal deletions were isolated that affect either the assembly of preinitiation complexes (PICs), scanning for AUG, or both. We show that eIF5 binds to the extreme c/Nip1-NTD (residues 1–45) and that impairing this interaction predominantly affects the PIC formation. eIF1 interacts with the region (60–137) that immediately follows, and altering this contact deregulates AUG recognition. Together, our data indicate that binding of eIF1 to the c/Nip1-NTD is equally important for its initial recruitment to PICs and for its proper functioning in selecting the translational start site.