Pandiarajan Ilamathy, Walunj Sujata B, Banerjee Nirjhar, Rout Janmejaya, Srivastava Sanjeeva, Patankar Swati, Kaledhonkar Sandip
Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India.
Front Mol Biosci. 2024 Aug 1;11:1398964. doi: 10.3389/fmolb.2024.1398964. eCollection 2024.
The ribosome, a ribonucleoprotein complex, performs the function of protein translation. While ribosomal RNA catalyzes polypeptide formation, several proteins assist the ribosome throughout the translation process. Studying the biochemical and kinetic properties of these proteins interacting with the ribosome is vital for elucidating their roles. Various techniques, such as zonal centrifugation, pull-down assays, dynamic light scattering (DLS), fluorescence polarization, and surface plasmon resonance (SPR) are employed for this purpose, each presenting unique advantages and limitations. We add to the repertoire of techniques by using Bio-Layer Interferometry (BLI) to examine interactions between the ribosome and translation factors. Our findings demonstrate that BLI can detect interactions of ribosomes with two proteins: initiation factor 2 (IF2) and translation enhancing factor (PTEF). A protein (Green Fluorescent Protein; GFP) known not to bind to ribosomes, shows no binding in the BLI assay. We show that BLI could be used to study the ribosome-protein interactions as it has key advantages like label-free procedures, ease of assay performance, and ribosome sample reuse. Our results highlight the comprehensive use of BLI in studying the ribosome-protein interactions, in addition to studying protein-protein and protein-ligand interactions.
核糖体是一种核糖核蛋白复合体，执行蛋白质翻译功能。虽然核糖体RNA催化多肽形成，但在整个翻译过程中有几种蛋白质协助核糖体。研究这些与核糖体相互作用的蛋白质的生化和动力学特性对于阐明它们的作用至关重要。为此采用了各种技术，如区带离心、下拉分析、动态光散射（DLS）、荧光偏振和表面等离子体共振（SPR），每种技术都有独特的优点和局限性。我们通过使用生物层干涉术（BLI）来研究核糖体与翻译因子之间的相互作用，从而增加了技术手段。我们的研究结果表明，BLI可以检测核糖体与两种蛋白质的相互作用：起始因子2（IF2）和翻译增强因子（PTEF）。一种已知不与核糖体结合的蛋白质（绿色荧光蛋白；GFP）在BLI分析中未显示结合。我们表明，BLI可用于研究核糖体 - 蛋白质相互作用，因为它具有无标记程序、易于进行分析和核糖体样品可重复使用等关键优点。我们的结果突出了BLI在研究核糖体 - 蛋白质相互作用以及蛋白质 - 蛋白质和蛋白质 - 配体相互作用方面的综合应用。
