Thanedar S, Kumar N V, Varshney U
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
J Biol Chem. 2000 Jul 7;275(27):20361-7. doi: 10.1074/jbc.M001238200.
Formylation of the initiator tRNA is essential for normal growth of Escherichia coli. The initiator tRNA containing the U35A36 mutation (CUA anticodon) initiates from UAG codon. However, an additional mutation at position 72 (72A --> G) renders the tRNA (G72/U35A36) inactive in initiation because it is defective in formylation. In this study, we isolated U1G72/U35A36 tRNA containing a wobble base pair at 1-72 positions as an intragenic suppressor of the G72 mutation. The U1G72/U35A36 tRNA is formylated and participates in initiation. More importantly, we show that the mismatch at 1-72 positions of the initiator tRNA, which was thus far thought to be the hallmark of the resistance of this tRNA against peptidyl-tRNA hydrolase (PTH), is not sufficient. The amino acid attached to the initiator tRNA is also important in conferring protection against PTH. Further, we show that the relative levels of PTH and IF2 influence the path adopted by the initiator tRNAs in protein synthesis. These findings provide an important clue to understand the dual function of the single tRNA(Met) in initiation and elongation, in the mitochondria of various organisms.
起始tRNA的甲酰化对于大肠杆菌的正常生长至关重要。含有U35A36突变（反密码子为CUA）的起始tRNA从UAG密码子起始。然而，第72位的另一个突变（72A→G）使tRNA（G72/U35A36）在起始过程中失活，因为它在甲酰化方面存在缺陷。在本研究中，我们分离出了在1-72位含有摆动碱基对的U1G72/U35A36 tRNA，作为G72突变的基因内抑制子。U1G72/U35A36 tRNA被甲酰化并参与起始过程。更重要的是，我们发现起始tRNA在1-72位的错配，迄今为止一直被认为是该tRNA对肽基-tRNA水解酶（PTH）抗性的标志，但这是不够的。连接在起始tRNA上的氨基酸在赋予对PTH的抗性方面也很重要。此外，我们表明PTH和IF2的相对水平会影响起始tRNA在蛋白质合成中所采用的途径。这些发现为理解各种生物体线粒体中单个tRNA(Met)在起始和延伸中的双重功能提供了重要线索。
