Translation initiation without IF2-dependent GTP hydrolysis.
Translation initiation factor IF2 is a guanine nucleotide-binding protein. The free energy change associated with guanosine triphosphate hydrolase (GTPase) activity of these proteins is believed to be the driving force allowing them to perform their functions as molecular switches. We examined role and relevance of IF2 GTPase and demonstrate that an Escherichia coli IF2 mutant bearing a single amino acid substitution (E571K) in its 30S binding domain (IF2-G3) can perform in vitro all individual translation initiation functions of wild type (wt) IF2 and supports faithful messenger RNA translation, despite having a reduced affinity for the 30S subunit and being completely inactive in GTP hydrolysis. Furthermore, the corresponding GTPase-null mutant of Bacillus stearothermophilus (E424K) can replace in vivo wt IF2 allowing an E. coli infB null mutant to grow with almost wt duplication times. Following the E571K (and E424K) mutation, which likely disrupts hydrogen bonding between subdomains G2 and G3, IF2 acquires a guanosine diphosphate (GDP)-like conformation, no longer responsive to GTP binding thereby highlighting the importance of interdomain communication in IF2. Our data underlie the importance of GTP as an IF2 ligand in the early initiation steps and the dispensability of the free energy generated by the IF2 GTPase in the late events of the translation initiation pathway.
Laboratory of Genetics, Department of Biosciences and Biotechnology, University of Camerino, 62032 Camerino, Macerata, Italy.
Attilio
A
Fabbretti
Letizia
L
Brandi
Pohl
P
Milón
Roberto
R
Spurio
Cynthia L
CL
Pon
Claudio O
CO
Gualerzi
D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D009154:Mutation; D010442:Peptide Chain Initiation, Translational; D039881:Prokaryotic Initiation Factor-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D054680:Ribosome Subunits, Small, Bacterial / Q000378:metabolism
D039881:Prokaryotic Initiation Factor-2; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases