Effect of the Amino Acid Attached to Escherichia coli Initiator tRNA on Its Affinity for the Initiation Factor IF2 and on the IF2 Dependence of Its Binding to the Ribosome

Abstract

We show that the nature of the amino acid in the formylaminoacyl-tRNA influences initiation factor (IF) 2 dependence of its ribosome binding and that this IF2 dependence reflects the relative affinity of the formylaminoacyl-tRNA for the initiation factor IF2. We compared the template-dependent ribosome binding activities, in the presence of initiation factors, of wild type and anticodon sequence mutants of Escherichia coli initiator tRNAs that carry formylmethionine (fMet), formylglutamine (fGln), or formylvaline (fVal). The fGlntRNA bound less well than fMet-tRNA whereas the fValtRNA bound as well as fMet-tRNA. The rate and extent of binding of fGln-tRNA to the ribosome was significantly increased by further addition of purified initiation factor IF2. In contrast, the binding of fVal-tRNA or fMettRNA was not affected much by the addition of IF2. Using gel mobility shift assay, we have measured the apparent K d values of the IF2⅐formylaminoacyl-tRNA binary complexes. These are 1.8, 3.5, and 10.5 M for fMettRNA, fVal-tRNA, and fGln-tRNA, respectively.Two classes of methionine tRNAs are present in all organisms (reviewed in Refs. 1-4). One of these, the initiator, is used for initiation of protein synthesis whereas the other, the elongator, is used for insertion of methionine internally. In eubacteria and in eukaryotic organelles such as mitochondria and chloroplasts, the initiator tRNA is used as formylmethionyltRNA (fMet-tRNA). The fMet-tRNA binds to the P site on the ribosome, whereas the elongator tRNAs bind to the A site. Binding of the fMet-tRNA to the P site is facilitated by the initiation factors IF1, 1 IF2, and IF3 in Escherichia coli (5). IF2 is thought to help the ribosome select fMet-tRNA over other tRNAs by virtue of the fact that, outside of the peptidyl-tRNAs on the ribosome, the fMet-tRNA is the only tRNA in the cell that carries an N-acyl-amino acid (6). However, the mechanism by which IF2 helps the ribosome achieve this selection is not clear.Results of in vitro studies originally led to the suggestion that IF2 acts as a carrier of fMet-tRNA to the ribosome in much the same way as the elongation factor EF-Tu (or eEF-1 in the case of eukaryotes) does for aminoacyl-tRNAs and the eukaryotic initiation factor elF-2 does for the corresponding initiator methionyl-tRNA (reviewed in Ref. 1). However, although IF2 and fMet-tRNA can form a specific binary complex, this complex is weak and dissociates readily in the presence of magnesium ions (7-9). Based on the estimated concentrations of IF2 in cells and the affinity of IF2 for the 30 S ribosome in the presence of the other initiation factors, it has been suggested that all of the 30 S ribosomal subunits are saturated with IF2 in vivo (5, 10). This and other in vitro results indicating that IF2 stimulated the binding also of elongator aminoacyl-tRNAs to the 30 S ribosome without any evidence for the formation of a binary complex between aminoacyl-tRNA and IF2 has led to the proposal that IF2 works at the ribosomal level by binding first to the 30 S subun...

Initiation of Protein Synthesis in Bacteria

“…The nature of the amino acid attached to the tRNA is less important for IF2 binding than is formylation. Hence, IF2 is able to bind to fVal-tRNA and fGln-tRNA but not to the unformylated tRNAs (251). However, moderate overexpression of IF2 leads to translation initiation without formylation of Met-tRNA f Met , and IF2 stimulates the binding of unformylated Met-tRNA f Met to 30S ribosomal subunits in vitro (68,250).…”

“…Original proposals suggested that IF2 carries fMet-tRNA f Met to the ribosome by analogy to EF1A for aminoacyl-tRNAs and eIF2 for Met-tRNA i Met (reviewed in reference 94). A specific binary complex can be formed between fMet-tRNA f Met and IF2 in vitro, but the interaction is weak, with a K d of 1.8 M, and the complex dissociates readily in the presence of magnesium ions (113,118,119,167,251). Most evidence suggests that IF2 performs its interactions with fMet-tRNA f Met on the 30S ribosomal subunit (251).…”

Initiation of Protein Synthesis in Mammalian Cells with Codons Other Than AUG and Amino Acids Other Than Methionine

“…Total tRNA was isolated from CV1 cells infected with SV40 carrying the G34C36 mutant initiator tRNA gene as described previously (10). Aminoacylation of tRNA with valine was performed using an E. coli S-100 extract enriched for ValRS as described by Wu and RajBhandary (57).…”

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

“…In vitro evidence suggests that the 30S subunit binds to mRNA or to fMet-tRNA in a random order and in rapid equilibrium to form a 30S-mRNAfMet-tRNA ternary complex, followed by a rate-limiting 'rearrangement' to form the 30S 'initiation complex' (Gualerzi and Pon, 1990). However, in vivo evidence suggests that, at least for some mRNAs, the 30S subunit binds first to the fMet-tRNA, delivered by IF2, and then binds the mRNA Wu and RajBhandary, 1997). The SD sequence, within the untranslated leader, provides for an increase in the K a of 30S-mRNA binary complex formation, but does not influence mechanistically the formation of the 'initiation complex', the rate of initiation, or the establishment of reading frame (Gualerzi and Pon, 1990).…”