The interdomain linker of Escherichia coli initiation factor IF3: a possible trigger of translation initiation specificity.
Initiation factor IF3 is responsible for the accuracy of translation initiation in bacteria, by destabilizing complexes involving non-initiator tRNA and/or nonstart codons. This proofreading is performed on the 30S subunit to which IF3 binds selectively. IF3 has an unusual architecture, with two globular domains connected by a mobile, positively charged linker. Here, we have investigated the function of this flexible tether by probing its conformation when IF3 is bound to the ribosomal RNA. Using site-directed mutagenesis of the linker region, we have also selectively modified its length, its flexibility and its chemical composition. The function of the mutant genes was assayed in vivo, and the structural and biochemical properties of some of the corresponding variant proteins were characterized in vitro. The two isolated domains of IF3 were also co-expressed in order to test the requirement for their covalent attachment. The results indicate that the physical link between the two domains of IF3 is essential for the function of this protein, but that the exact length and chemical composition of the linker can be varied to a large extent. A model is presented in which the extended linker would act as a 'strap', triggering a conformational change in the 30S subunit, which would then ensure initiator tRNA selection.
de Cock
E
E
Laboratoire de Biochimie, UMR 7654 du CNRS, Palaiseau, France.
Springer
M
M
Dardel
F
F
D019921:Biotinylation; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics*; D005816:Genetic Complementation Test; D009682:Magnetic Resonance Spectroscopy; D016254:Mutagenesis, Insertional; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000502:physiology*; D010957:Plasmids; D039665:Prokaryotic Initiation Factor-3; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism
D010448:Peptide Initiation Factors; D039665:Prokaryotic Initiation Factor-3; D012335:RNA, Ribosomal; D014357:Trypsin