Abstract
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 different 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 different 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.
Highlights
Translation initiation factor IF2 is a multi-domain GTPase playing essential roles in the initiation pathway of protein synthesis
IF2 binds with high affinity to the 30S ribosomal subunit with its N-terminal domain [9,10,11,12,13,14] and subsequently establishes a functional interaction with the ribosome [15,16], mainly through its G2 and G3 domains [12,13,17,18,19,20,21,22]. 30S-bound IF2 recruits the initiator fMet-tRNA [23] which is held in place on the subunit by an interaction between its acceptor end and the C-terminal domain C-2 of the factor [24,25,26] and by pairing of the anticodon bases with the mRNA initiation codon at a “pre-P-site” of the subunit [9,18,19,20,21,27]
The failure to express these mutant proteins was accompanied by a substantial decrease of the synthesis of the bulk cellular proteins, as seen from the result of the pulse-chase experiment in which the incorporation of [35S] methionine was compared in cells induced to express either wtIF2 or His301 mutants
Summary
Translation initiation factor IF2 is a multi-domain GTPase playing essential roles in the initiation pathway of protein synthesis (for reviews see [1,2,3,4,5,6,7,8,9]). 30S-bound IF2 recruits the initiator fMet-tRNA [23] which is held in place on the subunit by an interaction between its acceptor end and the C-terminal domain C-2 of the factor [24,25,26] and by pairing of the anticodon bases with the mRNA initiation codon at a “pre-P-site” of the subunit [9,18,19,20,21,27] During these early steps, a GTP molecule is bound to the G2 domain [17,18,28,29] conferring upon IF2 a “GTP conformation” [18,19,20,21,22,29,30,31]. IF2 eventually promotes the adjustment of the acceptor end of initiator fMet-tRNA in the productive P site of the 50S subunit so that the initiation dipeptide can be formed with the amino acid carried to the A site by an aminoacyl-tRNAEF-TuGTP ternary complex [32,37,38,39]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
