The nucleotide-binding site of bacterial translation initiation factor 2 (IF2) as a metabolic sensor

Abstract

Translational initiation factor 2 (IF2) is a guanine nucleotidebinding protein that can bind guanosine 3 ,5 -(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands. ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active protein syntheses and an inactive ppGpp-bound form when shortage of nutrients would be detrimental, if not accompanied by slackening of this synthesis.fast kinetics ͉ GTP ͉ translation regulation ͉ nutritional stress I nitiation factor 2 (IF2) is the only initiation factor that is ribosome-bound throughout the entire translation initiation pathway, participating initially in the formation of the 30S initiation complex (30SIC) and subsequently in the assembly of the 70S initiation complex (70SIC), a process that ultimately results in formation of the first peptide bond (initiation dipeptide) and generates the first ribosomal pretranslocation complex (for reviews, see refs. 1-5). Thus, it could be predicted that IF2 functions are accompanied͞modulated by conformational changes that could be either consequence or cause of the interactions of IF2 with its various ligands (30S and 50S ribosomal subunits, fMet-tRNA, GTP, GDP⅐Pi, and GDP). Crystallographic (6) and NMR (7) studies have, in fact, shown that, depending upon the nature of their ligand (i.e., GTP or GDP), aIF5B, the archaeal homologue of bacterial IF2, as well as isolated IF2G2, the G domain of IF2, undergo large structural changes. On the other hand, chemical probing (8) and cryo-EM (9, 10) have clearly shown that several conformational changes of the factor occur during the early, middle, and late events of translation initiation.Bacterial cells growing under optimal nutritional conditions contain a high (Ͼ1 mM) concentration of GTP and a vanishingly low level of GDP. Thus, IF2 is expected to exist and to bind the 30S subunit almost exclusively in the GTP form, because it displays similar affinity for GTP and GDP, both K d s being in the 10-to 100-M range (11). The IF2⅐GTP was shown to have a higher affinity for the 30S ribosomal subunit than either IF2⅐GDP or free IF2 (11). The adjustment of fMet-tRNA in the P site (ref. 12 and refs. therein) and the release of IF2 from 70SIC (13-15) have been attributed to the IF2-dependent GTP hydrolysis, which is very rapidly triggered by th...

Structure of BipA in GTP form bound to the ratcheted ribosome

“…S4F). Alike, ppGpp binding to IF2 interfers with binding to its interaction partners (41). This finding could provide a structural insight for the observation that BipA associates with either the 70S ribosome or the 30S subunit depending on the relative intracellular abundance of GTP and ppGpp (12).…”

ppGpp regulation of RpoS degradation via anti-adaptor protein IraP

“…Thus, in cells subjected to serine hydroxamate, significantly less IraP protein was detected than in cells subject to phosphate starvation. Whether this reflects a specific defect in IraP translation or increased IraP instability under these conditions (high ppGpp via RelA), or reflects the general inhibition of translation previously described for conditions of high ppGpp (23,32) has not been addressed. However, these results suggest that moderate levels of ppGpp (high enough to stimulate transcription but not high enough to inhibit translation) are the signal that is needed for invoking IraP stabilization of S .…”

Thermodynamic and Kinetic Framework of Selenocysteyl-tRNASec Recognition by Elongation Factor SelB

“…The nucleotide-binding properties of SelB (10) differ markedly from those of EF-Tu (11) and rather resemble those of EF-G or IF2 (12,13). SelB from Escherichia coli binds GTP and GDP with affinities of 0.7 M and 13 M, respectively (10).…”