Translation initiation factor IF1 of Bacillus stearothermophilus and Thermus thermophilus substitute for Escherichia coli IF1 in vivo and in vitro without a direct IF1–IF2 interaction

Kapralou, Stavroula; Fabbretti, Attilio; Garulli, Chiara; Spurio, Roberto; Gualerzi, Claudio O.; Dahĺberg, Albert E.; Pon, Cynthia L.

doi:10.1111/j.1365-2958.2008.06466.x

Cited by 13 publications

(11 citation statements)

References 37 publications

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“…The present results suggest that the stimulatory effect of IF1 may be mediated by a direct contact with the NTD of IF2. In contrast, thermophilic IF1 does not interact with the NTD of IF2 and does not augment IF2 functions [54]; consistently, no direct contact between IF1 and IF2 was found in the T. thermophilus 30S IC reconstruction [11]. The NTD region is significantly shorter in IF2 from T. thermophilus compared to E. coli , suggesting that the IF1–IF2 interaction is not universally conserved [54].…”

Section: Discussionmentioning

confidence: 91%

The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli

et al. 2011

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Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNAfMet requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNAfMet. Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNAfMet, IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNAfMet, which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNAfMet induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation.

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Section: Discussionmentioning

confidence: 91%

The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli

et al. 2011

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“…Furthermore, unlike domain IV of eIF5B, which binds eIF1A, IF2-C2 does not contain the C-terminal helices H13 and H14 responsible for the eIF1A–eIF5B interaction and binds instead to the initiator tRNA; also doubtful is the occurrence of an IF2-IF1 contact on the ribosome (39). Finally, although the eukaryotic suppressor mutations affect directly the ribosomal binding of eIF5B but do not affect its GTPase activity, the E571K and E424K likely represent ‘conformational’ mutants, which cause the complete inactivation of the GTPase activity of IF2 and affect indirectly its interaction with the ribosome.…”

Section: Discussionmentioning

confidence: 99%

Translation initiation without IF2-dependent GTP hydrolysis

Fabbretti

Brandi

Milón

et al. 2012

Nucleic Acids Research

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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.

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“…The precise IF2 topology derived from the present data and refined as a full-length model by MM simulations (Experimental Procedures) allows a detailed interpretation of the cryo-EM maps of the 30S and 70S ICs. The structure shows the presence of a kink around Pro355 in helix H8, close to where S12 and IF1 are bound on the 30S subunit; this proximity suggests that directly or indirectly, and without significantly affecting the thermodynamic stability of the IF2-ribosome complex (34), S12 (and part of IF1) may contribute to positioning domains C1 and C2 in such a way that interactions can be established with the fMet-tRNA fMet on the 30S IC. The structure reveals that in the functional 30S context IF2 adopts a well-defined, extended conformation; furthermore, the C1 and C2 domains are rather flexible in solution, in the crystal structure, or on the 70S IC after release of the interactions with the fMet-tRNA fMet (unless the N domain is removed, in which case these domains remain positioned like in the 30S IC and thereby prevent correct fMettRNA fMet binding in the P site, which is consistent with reduced dipeptide formation).…”

Section: Discussionmentioning

confidence: 99%

Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor

Simonetti

Marzi

Billas

et al. 2013

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

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Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/ L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl (fMet)-tRNA fMet positioning and efficient transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process.protein synthesis | integrated structural biology I n bacteria, three translation initiation factors (IF1, IF2, and IF3) participate in the first phase of protein synthesis during which the 30S subunit binds the initiator tRNA to form codonanticodon interactions with the mRNA. The 30S initiation complex (IC) thus formed is converted into a 70S IC by joining of the 50S ribosomal subunit (1, 2). The IFs enhance the rate of ICs formation and ensure translation accuracy. Within this process, IF2 plays a key role by promoting the recruitment and stabilization of the N-formyl-methionyl(fMet)-tRNA fMet on the 30S IC in the peptidyl/initiator (P/I) site of the small ribosomal subunit with the help of initiation factors IF1 and IF3 (3-6). IF2 mediates subunit joining on which its GTPase activity is stimulated, leading to the formation of a 70S IC in a conformation that is productive in the first transpeptidation reaction (7). Snapshots of this process, visualized by cryo electron microscopy (cryo-EM) (3,(8)(9)(10), have localized IF2 on the ribosome and provided structural insights into 30S and 70S IC's.Bacterial IF2 is a multidomain protein (Figs. S1 and S2) for which the overall architecture is unknown. Unlike 70S-interacting elongation GTPases, IF2 contains a specific N-terminal (N) domain that is variable in sequence (in Escherichia coli, two copies of it comprise the bona fide N domain and the G1 domain). Furthermore, it contains a highly conserved C-terminal region with a central core comprising domains G [GTP/GDP (G) binding domain, G2] and G3 (II), and a C-terminal part consisting of domain C1 and of the initiator tRNA-binding domain C2. Recently, the crystal structure of the core part (1-363) of Thermus thermophilus IF2 was determined in different functional states (apo-protein, GTP, and GDP forms) (11). These structures gave important insights into the mechanism of nucleotide binding and GTP hydrolysis and also reveal significant structural differences with a related archaeal and eukaryotic protein a/eIF5B (11). However, because the entire C-terminal region was ...

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Translation initiation factor IF1 of Bacillus stearothermophilus and Thermus thermophilus substitute for Escherichia coli IF1 in vivo and in vitro without a direct IF1–IF2 interaction

Cited by 13 publications

References 37 publications

The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli

The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli

Translation initiation without IF2-dependent GTP hydrolysis

Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor

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