Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA.
Mol Cell Biol. 2010 Sep;30(18):4415-34. doi: 10.1128/MCB.00280-10. Epub 2010 Jun 28.
The C-terminal domain (CTD) of the a/Tif32 subunit of budding yeast eukaryotic translation initiation factor 3 (eIF3) interacts with eIF3 subunits j/Hcr1 and b/Prt1 and can bind helices 16 to 18 of 18S rRNA, suggesting proximity to the mRNA entry channel of the 40S subunit. We have identified substitutions in the conserved Lys-Glu-Arg-Arg (KERR) motif and in residues of the nearby box6 element of the a/Tif32 CTD that impair mRNA recruitment by 43S preinitiation complexes (PICs) and confer phenotypes indicating defects in scanning and start codon recognition. The normally dispensable CTD of j/Hcr1 is required for its binding to a/Tif32 and to mitigate the growth defects of these a/Tif32 mutants, indicating physical and functional interactions between these two domains. The a/Tif32 CTD and the j/Hcr1 N-terminal domain (NTD) also interact with the RNA recognition motif (RRM) in b/Prt1, and mutations in both subunits that disrupt their interactions with the RRM increase leaky scanning of an AUG codon. These results, and our demonstration that the extreme CTD of a/Tif32 binds to Rps2 and Rps3, lead us to propose that the a/Tif32 CTD directly stabilizes 43S subunit-mRNA interaction and that the b/Prt1-RRM-j/Hcr1-a/Tif32-CTD module binds near the mRNA entry channel and regulates the transition between scanning-conducive and initiation-competent conformations of the PIC.
酵母真核翻译起始因子 3(eIF3)的 a/Tif32 亚基的 C 端结构域(CTD)与 eIF3 亚基 j/Hcr1 和 b/Prt1 相互作用,并且可以与 18S rRNA 的 16 到 18 号螺旋结合,提示其与 40S 亚基的 mRNA 进入通道接近。我们已经鉴定出 a/Tif32 CTD 中的保守赖氨酸-谷氨酸-精氨酸-精氨酸(KERR)基序和附近盒 6 元件中的残基的取代,这些取代会损害 43S 起始前复合物(PIC)对 mRNA 的招募,并赋予表明扫描和起始密码子识别缺陷的表型。j/Hcr1 的通常非必需的 CTD 对于其与 a/Tif32 的结合以及减轻这些 a/Tif32 突变体的生长缺陷是必需的,这表明这两个结构域之间存在物理和功能相互作用。a/Tif32 CTD 和 j/Hcr1 N 端结构域(NTD)也与 b/Prt1 中的 RNA 识别基序(RRM)相互作用,并且破坏其与 RRM 相互作用的这两个亚基中的突变增加了 AUG 密码子的渗漏扫描。这些结果以及我们证明 a/Tif32 的极端 CTD 与 Rps2 和 Rps3 结合,使我们提出 a/Tif32 CTD 直接稳定 43S 亚基-mRNA 相互作用,并且 b/Prt1-RRM-j/Hcr1-a/Tif32-CTD 模块结合在 mRNA 进入通道附近并调节 PIC 之间的扫描诱导和起始能力之间的转变。