Kropiwnicka Anna, Kuchta Krzysztof, Lukaszewicz Maciej, Kowalska Joanna, Jemielity Jacek, Ginalski Krzysztof, Darzynkiewicz Edward, Zuberek Joanna
College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland; Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland.
College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland; Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland.
Biochem Biophys Res Commun. 2015 Jan 2;456(1):47-52. doi: 10.1016/j.bbrc.2014.11.032. Epub 2014 Nov 18.
The assembly of the ribosome on majority of eukaryotic mRNAs is initiated by the recruitment of eIF4E protein to the mRNA 5' end cap structure. Flowering plants use two eIF4E isoforms, named eIF4E and eIF(iso)4E, as canonical translation initiation factors and possess a homolog of mammalian 4EHP (or eIF4E-2) termed nCBP. Plants from Brassicaceae family additionally conserve a close paralog of eIF4E which in Arabidopsis thaliana has two copies named eIF4E1b and eIF4E1c. In order to assess the efficiency of plant non-canonical (eIF4E1b/1c and nCBP) and canonical (eIF4E and eIF(iso)4E) eIF4E proteins to bind mRNAs we utilized fluorescence titrations to determine accurate binding affinities of five A.thaliana eIF4E isoforms for a series of cap analogs. We found that eIF4E binds cap analogs from 4-fold to 10-fold stronger than eIF(iso)4E, while binding affinities of nCBP and eIF(iso)4E are comparable. Furthermore, eIF4E1c interacts similarly strongly with the cap as eIF4E, but eIF4E1b binds cap analogs ca. 2-fold weaker than eIF4E1c, regardless of the 95% sequence identity between these two proteins. The use of differentially chemically modified cap analogs in binding studies and a detailed analysis of the obtained homology models gave us insight into the molecular characteristic of varying cap-binding abilities of Arabidopsis eIF4E isoforms.
大多数真核生物mRNA上核糖体的组装是通过将eIF4E蛋白募集到mRNA 5'端帽结构上开始的。开花植物使用两种eIF4E亚型，即eIF4E和eIF(iso)4E，作为典型的翻译起始因子，并拥有哺乳动物4EHP（或eIF4E-2）的同源物，称为nCBP。十字花科植物还保留了eIF4E的一个紧密旁系同源物，在拟南芥中有两个拷贝，分别命名为eIF4E1b和eIF4E1c。为了评估植物非典型（eIF4E1b/1c和nCBP）和典型（eIF4E和eIF(iso)4E）eIF4E蛋白与mRNA结合的效率，我们利用荧光滴定法来确定拟南芥五种eIF4E亚型与一系列帽类似物的准确结合亲和力。我们发现，eIF4E与帽类似物的结合强度比eIF(iso)4E强4至10倍，而nCBP和eIF(iso)4E的结合亲和力相当。此外，eIF4E1c与帽的相互作用强度与eIF4E相似，但eIF4E1b与帽类似物的结合比eIF4E1c弱约2倍，尽管这两种蛋白之间有95%的序列同一性。在结合研究中使用化学修饰不同的帽类似物以及对所得同源模型的详细分析，使我们深入了解了拟南芥eIF4E亚型不同帽结合能力的分子特征。
