Proteolysis of <i>Bacillus stearothermophilus</i> IF2 and specific protection by GTP

Severini, M.; Choli, Theodora; Teana, Anna La; Gualerzi, Claudio O.

doi:10.1016/0014-5793(90)80495-5

Cited by 15 publications

(21 citation statements)

References 9 publications

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“…1). In the absence of fMet-tRNA, the digestion pattern is virtually identical to that previously reported [7] as also confirmed by the peptide sequences determined (see below). In the presence of the initiator tRNA, on the other hand, the proteolysis yields some characteristic peptides of higher molecular weight which renmin un- • .…”

Section: Resultssupporting

confidence: 87%

“…The binding site for fMet-tRNA has been localized in the 24.5 kDa carboxyl-terminal portion of the protein (the C-domain) which appears to be very compact and resistant to proteolytic attack; the 50 S binding site, the GTP/GDP binding site, the GTPase catalytic center and part of the 30 S binding site have been localized in the central domain of the protein, the G-domain of approximately 41 kDa [4,5]. The interaction of IF2 with GTP or GDP protects the G-domain against a proteolytic cleavage occurring at a specific site (Arg~°S-Ala 3°9) localized within the GTP/GDP binding site [6,7]. The N-terminal portion of the protein, on the other hand, probably has a floppy structure which is very susceptible to proteolytic attack; this part of the protein displays a nonconserved sequence and does not seem to be implicated in any fundamental translational function [4,8,9] and its role remains obscure.…”

Section: Introductionmentioning

confidence: 99%

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Proteolysis of Bacillus stearothermophilus IF2 and specific protection by fMet‐tRNA

et al. 1992

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Translation initiation factor IF2 from Bacillus stearothermophilus (741 amino acids, M r 82,043) was subjected to trypsinolysis alone or in the presence of fMet-tRNA. The initiator tRNA was found to protect very efficiently the Arg3°S-Ala 3~ bond within the GTP binding site of IF2 and, more weakly, three bonds (Lys~6-Gln~47, LystS~-Glu ~ss and ArgS~-SerS~°). The first two are located at the border between the non-conserved, dispen~ble (for translation) N-terminal portion and the conserved G-domain of the protein, the third is located at the border between the G-and C.domains. Since IF2 is known to interact with fMet-tRNA through its protease.resistant C-(earboxyl terminus) domain, the observed protection suggests that, upon binding of fMet.tRNA, long-distance tertiary interactions between the IF2 domains may take place.

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Section: Resultssupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Proteolysis of Bacillus stearothermophilus IF2 and specific protection by fMet‐tRNA

et al. 1992

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“…2,9 The functional complexity of IF2 is paralleled by a comparable structural complexity. 9 Early analyses of the domain structure of IF2 carried out by a functional characterization of the proteolytic fragments of Escherichia coli 10 and Bacillus stearothermophilus [11][12][13] IF2 obtained in the presence and absence of various ligands have allowed us to identify three large regions, the Nterminal domain, a large (41 kDa) central domain containing the conserved GTP binding elements (hence denominated G-domain) and the C-terminal domain (see Figure 1). In turn, in the absence of GTP or GDP, the G-domain was further cleaved by proteolytic enzymes, yielding two fragments of similar size originally designated GI and GII.…”

Section: Introductionmentioning

confidence: 99%

Translation Initiation Factor IF2 Interacts with the 30 S Ribosomal Subunit via Two Separate Binding Sites

Caserta

Tomšič

Spurio

et al. 2006

Journal of Molecular Biology

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“…Like the other GTP-binding proteins involved in translation (EF-Tu, EF-G, and RF3), IF2 contains a guanine nucleotide-binding site that can bind GTP and GDP, as well as ppGpp (25)(26)(27)(28).…”

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confidence: 99%

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

Milón

Tischenko²,

Tomšič³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

166

186

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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...

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Proteolysis of Bacillus stearothermophilus IF2 and specific protection by GTP

Cited by 15 publications

References 9 publications

Proteolysis of Bacillus stearothermophilus IF2 and specific protection by fMet‐tRNA

Proteolysis of Bacillus stearothermophilus IF2 and specific protection by fMet‐tRNA

Translation Initiation Factor IF2 Interacts with the 30 S Ribosomal Subunit via Two Separate Binding Sites

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

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