Ayyub Shreya Ahana, Dobriyal Divya, Varshney Umesh
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
J Bacteriol. 2017 May 9;199(11). doi: 10.1128/JB.00051-17. Print 2017 Jun 1.
Initiation factor 3 (IF3) is one of the three conserved prokaryotic translation initiation factors essential for protein synthesis and cellular survival. Bacterial IF3 is composed of a conserved architecture of globular N- and C-terminal domains (NTD and CTD) joined by a linker region. IF3 is a ribosome antiassociation factor which also modulates selection of start codon and initiator tRNA. All the functions of IF3 have been attributed to its CTD by studies. However, the relevance of these findings has not been investigated. By generating complete and partial IF3 () knockouts in and by complementation analyses using various deletion constructs, we show that while the CTD is essential for survival, the NTD is not. Polysome profiles reaffirm that CTD alone can bind to the 30S ribosomal subunit and carry out the ribosome antiassociation function. Importantly, in the absence of the NTD, bacterial growth is compromised, indicating a role for the NTD in the fitness of cellular growth. Using reporter assays for initiation, we show that the NTD plays a crucial role in the fidelity function of IF3 by avoiding (i) initiation from non-AUG codons and (ii) initiation by initiator tRNAs lacking the three highly conserved consecutive GC pairs (in the anticodon stem) known to function in concert with IF3. Initiation factor 3 regulates the fidelity of eubacterial translation initiation by ensuring the formation of an initiation complex with an mRNA bearing a canonical start codon and with an initiator tRNA at the ribosomal P site. Additionally, IF3 prevents premature association of the 50S ribosomal subunit with the 30S preinitiation complex. The significance of our work in is in demonstrating that while the C-terminal domain alone sustains for its growth, the N-terminal domain adds to the fidelity of initiation of protein synthesis and to the fitness of the bacterial growth.
起始因子3（IF3）是三种保守的原核生物翻译起始因子之一，对蛋白质合成和细胞存活至关重要。细菌IF3由球状的N端和C端结构域（NTD和CTD）通过连接区相连的保守结构组成。IF3是一种核糖体抗结合因子，它还能调节起始密码子和起始tRNA的选择。此前的研究已将IF3的所有功能都归因于其CTD。然而，这些发现的相关性尚未得到研究。通过在大肠杆菌中构建完整和部分的IF3基因敲除株，并使用各种缺失构建体进行互补分析，我们发现虽然CTD对大肠杆菌的存活至关重要，但NTD并非如此。多核糖体图谱再次证实，单独的CTD就能与30S核糖体亚基结合并发挥核糖体抗结合功能。重要的是，在没有NTD的情况下，细菌生长受到影响，这表明NTD在细胞生长适应性方面发挥作用。通过使用报告基因检测起始过程，我们发现NTD在IF3的保真功能中起着关键作用，它可以避免：（i）从非AUG密码子起始；（ii）由缺乏与IF3协同作用的三个高度保守连续GC对（位于反密码子茎中）的起始tRNA起始。起始因子3通过确保与带有标准起始密码子的mRNA以及核糖体P位点上的起始tRNA形成起始复合物来调节真细菌翻译起始的保真度。此外，IF3可防止50S核糖体亚基与30S起始前复合物过早结合。我们在大肠杆菌中的工作意义在于表明，虽然单独的C端结构域能维持其生长，但N端结构域增加了蛋白质合成起始的保真度以及细菌生长的适应性。
