Site-directed mutagenesis of Escherichia coli translation initiation factor IF1. Identification of the amino acids involved in its ribosomal binding and recycling

Abstract

Starting from a synthetic modular gene (infA) encoding Escherichia coli translation initiation factor IF1, we have constructed mutants in which amino acids are deleted from the carboxyl terminus or in which His29 or His34 are replaced by Tyr or Asp residues. The mutant proteins were overproduced, purified and tested in vitro for their properties in several partial reactions of the translation initiation pathway and for their capacity to stimulate MS2 RNA-dependent protein synthesis. The results allow for the conclusion that: (i) Arg69 is part of the 30S ribosomal subunit binding site of IF1 and its deletion results in the substantial loss of all IF1 function; (ii) neither one of its two histidines is essential for the binding of IF1 to the 30S ribosomal subunit, for the stimulation of fMet-tRNA binding to 30S or 70S ribosomal particles or for MS2 RNA-dependent protein synthesis; but (iii) His29 is involved in the 50S subunit-induced ejection of IF1 from the 30S ribosomal subunit.

Crystal Structure of an Initiation Factor Bound to the 30 S Ribosomal Subunit

“…The abolition of IF1 binding in A1492G or A1493G mutants is consistent with the bulkier guanine base being unable to fit in either binding pocket (22). The increase in reactivity of A1408 (14) is explained by the disruption of the base pair it makes with A1493 in the native structure (16,23 (24), which is consistent with the observation that the equivalent residue in Thermus (Tyr 35 ) makes hydrogen bonding interactions with the phosphate backbone of C519. Further details of the IF1-30S interactions are described in the supplementary material (25).…”

In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level

“…The amino-acid residue Arg69 has been shown to be a part of the 30S binding site and deletion of the last three residues in IF1 (Arg69-Ser70-Arg71) results in substantially lowered affinity of IF1 for the 30S subunit [40]. In addition, this version of IF1 with the C-terminal deletion shows a much reduced biological activity in promoting 30S initiation complex formation.…”

Initiation of Protein Synthesis in Bacteria

“…In addition, C519 and G530 of the 530 loop and the amino acids V40 and W42 of ribosomal protein S12 are important for the interaction. A number of amino acids crucial for IF1 function were identified by site-directed mutagenesis (37,65,216). Moreover, specific signal changes in NMR spectroscopy experiments on titration of IF1 with 30S ribosomal subunits have been used to identify positions on IF1 involved in the interaction (204) (Fig.…”