Site-directed mutagenesis of Escherichia coli translation initiation factor IF1. Identification of the amino acid involved in its ribosomal binding and recycling.
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.
Gualerzi
C O
CO
Department of Cell Biology, University of Camerino, Italy.
Spurio
R
R
La Teana
A
A
Calogero
R
R
Celano
B
B
Pon
C L
CL
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics*; D015977:Eukaryotic Initiation Factor-1 / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D015202:Protein Engineering; D012270:Ribosomes / Q000378:metabolism*
D000596:Amino Acids; D015977:Eukaryotic Initiation Factor-1; D006639:Histidine