The Fate of the Initiator tRNAs Is Sensitive to the Critical Balance between Interacting Proteins

The Fate of the Initiator tRNAs Is Sensitive to the Critical Balance between Interacting Proteins

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Andrew Castleman The Fate of the Initiator tRNAs Is Sensitive to the Critical Balance between Interacting Proteins * Swapna Thanedar , N. Vinay Kumar and Umesh Varshney Abstract 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. Organisms have evolved with two distinct species of methionyl tRNAs. Of these, the initiator recognizes the initiation codons (AUG, GUG, AUU, UUG, etc.), and the elongator decodes the subsequent AUG codons in a mRNA ( 1 ). Both species of the tRNA are aminoacylated by the same methionyl-tRNA synthetase. In eubacteria, the initiators (Met-tRNA fMet ) are then further modified by formyltransferase to generate formylmethionyl-tRNA (fMet-tRNA fMet ), which interacts with IF2 to participate at the step of initiation ( 2-4 ). On the other hand, the elongators (Met-tRNA Met ) bind to EFTu to participate in elongation cycles. The major structural features that distinguish the initiators from elongators are located within the acceptor and the anticodon stems ( 5-7 ). A striking feature of the eubacterial initiator tRNAs is the presence of a mismatch at the 1–72 positions, which constitutes an important element for their recognition by formyltransferase ( 8-11 ). The mismatch also prevents the binding of the initiators to EF-Tu and renders the fMet-tRNA fMet resistant to peptidyl-tRNA hydrolase, PTH 1 ( 12 ), an enzyme which hydrolyzes N-blocked aminoacyl and the peptidyl tRNAs to facilitate tRNA recycling ( 13 , 14 ). The initiator tRNA containing CAU to CUA anticodon change, tRNA fMet (U35A36), is aminoacylated with glutamine ( 15 ) and initiates with formyl-glutamine utilizing UAG as an initiation codon in Escherichia coli ( 16 ). However, the tRNA
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The Fate of the Initiator tRNAs Is Sensitive to the Critical Balance between Interacting Proteins

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