Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India.
Nucleic Acids Res. 2018 Nov 30;46(21):11566-11574. doi: 10.1093/nar/gky859.
Living organisms possess two types of tRNAs for methionine. Initiator tRNAs bind directly into the ribosomal P-site to initiate protein synthesis, and the elongators bind to the A-site during the elongation step. Eubacterial initiators (tRNAfMet) are unique in that the methionine attached to them is formylated to facilitate their binding to initiation factor 2 (IF2), and to preclude them from binding to elongation factor Tu (EFTu). However, in mammalian mitochondria, protein synthesis proceeds with a single dual function tRNAMet. Escherichia coli possesses four tRNAfMet (initiator) and two tRNAMet (elongator) genes. Free-living organisms possessing the mitochondrion like system of single tRNAMet are unknown. We characterized mutants of E. coli tRNAfMet that function both as initiators and elongators. We show that some of the tRNAfMet mutants sustain E. coli lacking all four tRNAfMet and both tRNAMet genes, providing a basis for natural occurrence of mitochondria like situation in free living organisms. The tRNA mutants show in vivo binding to both IF2 and EFTu, indicating how they carry out these otherwise mutually exclusive functions by precise regulation of their in vivo formylation. Our results provide insights into how distinct initiator and elongator methionine tRNAs might have evolved from a single 'dual function' tRNA.
生物体内存在两种甲硫氨酸 tRNA。起始 tRNA 可直接与核糖体 P 位结合，起始蛋白质合成，延伸因子则在延伸步骤与 A 位结合。原核生物的起始 tRNA（tRNAfMet）较为独特，其携带的甲硫氨酸被甲酰化，以促进与起始因子 2（IF2）的结合，并阻止其与延伸因子 Tu（EFTu）结合。然而，在哺乳动物线粒体中，蛋白质合成使用单一的双功能 tRNAMet。大肠杆菌拥有四个 tRNAfMet（起始）和两个 tRNAMet（延伸）基因。目前尚未发现自由生活的生物体拥有类似于线粒体的单 tRNAMet 系统。我们对大肠杆菌 tRNAfMet 的突变体进行了研究，这些突变体能同时发挥起始和延伸作用。我们发现，一些 tRNAfMet 突变体能维持缺乏所有四个 tRNAfMet 和两个 tRNAMet 基因的大肠杆菌，为自由生活的生物体中存在类似线粒体的情况提供了自然发生的基础。这些 tRNA 突变体在体内与 IF2 和 EFTu 结合，表明它们如何通过对体内甲酰化的精确调节来执行这些原本相互排斥的功能。我们的研究结果为研究不同的起始和延伸甲硫氨酸 tRNA 如何从单个“双功能”tRNA 进化而来提供了线索。
