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

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 3 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 fMet (U35A36) was rendered nonfunctional in initiation when coupled with an A to G mutation at position 72 (G72/U35A36). The G72 mutation replaced the CxA mismatch at the top of the acceptor stem with a strong C-G base pair. The G72/U35A36 tRNA could be aminoacylated with glutamine but failed to initiate because it is an extremely poor substrate for formyltransferase (8,17).In this report, we show that a spontaneous intragenic mutation (C 3 T) corresponding ...

Initiation of Protein Synthesis in Bacteria

“…Therefore, the absence of a base pair between nucleotides 1 and 72 protects fMet-RNA f Met against hydrolytic cleavage by PTH. The amino acid attached to tRNA f Met is also important since fMettRNA f Met is completely resistant to hydrolysis by PTH whereas fGln-tRNA f Met is not (228). In most cases, the formyl group and the methionine residue are removed posttranslationally or even as the nascent polypeptide chain emerges from the ribosomal exit tunnel (94).…”

An unexpected absence of queuosine modification in the tRNAs of an Escherichia coli B strain

“…Isolation of tRNAs and Northern blot analysis. The tRNAs were isolated under cold and acidic conditions to preserve the ester linkage between the amino acid and tRNA, electrophoresed on 6n5 % polyacrylamide gels containing 8 M urea under acidic conditions (Varshney et al, 1991), electroblotted onto nytran membranes and hybridized (Thanedar et al, 2000) with 5h-$#P-end-labelled oligonucleotide. The probes were complementary to positions 29-47 of tRNA # fMet(CUA) , 2-37 of tRNA Tyr or 30-49 of tRNA His (Sprinzl et al, 1989).…”

Peptidyl-tRNA hydrolase and its critical role in protein biosynthesis

“…Notably, a mismatch at the top of the acceptor stem (positions 1 and 72), unique to the prokaryotic initiators, is a hallmark of their resistance to Pth (Kössel & RajBhandary, 1968;Schulman & Pelka, 1975;Dutka et al, 1993). The amino acid attached to the initiator tRNA fMet also contributes to its resistance to Pth (Thanedar et al, 2000).…”