Disparate Phenotypes Resulting from Mutations of a Single Histidine in Switch II of Geobacillus stearothermophilus Translation Initiation Factor IF2

The conserved Histidine 301 in switch II of Geobacillus stearothermophilus IF2 G2 domain was substituted with Ser, Gln, Arg, Leu and Tyr to generate mutants displaying different phenotypes. Overexpression of IF2H301S, IF2H301L and IF2H301Y in cells expressing wtIF2, unlike IF2H301Q and IF2H301R, cau...

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Veröffentlicht in:International journal of molecular sciences 2020-01, Vol.21 (3), p.735, Article 735
Hauptverfasser: Tomsic, Jerneja, Smorlesi, Arianna, Caserta, Enrico, Giuliodori, Anna Maria, Pon, Cynthia L., Gualerzi, Claudio O.
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Sprache:eng
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Zusammenfassung:The conserved Histidine 301 in switch II of Geobacillus stearothermophilus IF2 G2 domain was substituted with Ser, Gln, Arg, Leu and Tyr to generate mutants displaying different phenotypes. Overexpression of IF2H301S, IF2H301L and IF2H301Y in cells expressing wtIF2, unlike IF2H301Q and IF2H301R, caused a dominant lethal phenotype, inhibiting in vivo translation and drastically reducing cell viability. All mutants bound GTP but, except for IF2H301Q, were inactive in ribosome-dependent GTPase for different reasons. All mutants promoted 30S initiation complex (30S IC) formation with wild type (wt) efficiency but upon 30S IC association with the 50S subunit, the fMet-tRNA reacted with puromycin to di fferent extents depending upon the IF2 mutant present in the complex (wtIF2 >= to IF2H301Q > IF2H301R >>> IF2H301S, IF2H301L and IF2H301Y) whereas only fMet-tRNA 30S-bound with IF2H301Q retained some ability to form initiation dipeptide fMet-Phe. Unlike wtIF2, all mutants, regardless of their ability to hydrolyze GTP, displayed higher affinity for the ribosome and failed to dissociate from the ribosomes upon 50S docking to 30S IC. We conclude that di fferent amino acids substitutions of His301 cause different structural alterations of the factor, resulting in disparate phenotypes with no direct correlation existing between GTPase inactivation and IF2 failure to dissociate from ribosomes.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms21030735