Structural Dynamics of Bacterial Translation Initiation Factor IF2

Wienk, Hans; Tishchenko, E. V.; Belardinelli, Riccardo; Tomaselli, Simona; Dongre, Ramachandra; Spurio, Roberto; Folkers, Gert E.; Gualerzi, Claudio O.; Boelens, Rolf

doi:10.1074/jbc.m111.333393

Cited by 27 publications

(57 citation statements)

References 66 publications

(80 reference statements)

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“…NMR experiments and cryo-EM reconstructions support a model where IF2 is less flexible when interacting with ribosomal complexes (6,7,9,10,13). Cryo-EM reconstructions of 30S and 70S ICs with IF2 bound show the overall structure of IF2 when it is bound to the ribosome with some noticeable differences (6, 7, 9, 10).…”

Section: Discussionmentioning

confidence: 68%

See 1 more Smart Citation

Initiation factor 2 crystal structure reveals a different domain organization from eukaryotic initiation factor 5B and mechanism among translational GTPases

Eiler

Lin

Simonetti

et al. 2013

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

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The initiation of protein synthesis uses initiation factor 2 (IF2) in prokaryotes and a related protein named eukaryotic initiation factor 5B (eIF5B) in eukaryotes. IF2 is a GTPase that positions the initiator tRNA on the 30S ribosomal initiation complex and stimulates its assembly to the 50S ribosomal subunit to make the 70S ribosome. The 3.1-Å resolution X-ray crystal structures of the fulllength Thermus thermophilus apo IF2 and its complex with GDP presented here exhibit two different conformations (all of its domains except C2 domain are visible). Unlike all other translational GTPases, IF2 does not have an effecter domain that stably contacts the switch II region of the GTPase domain. The domain organization of IF2 is inconsistent with the "articulated lever" mechanism of communication between the GTPase and initiator tRNA binding domains that has been proposed for eIF5B. Previous cryo-electron microscopy reconstructions, NMR experiments, and this structure show that IF2 transitions from being flexible in solution to an extended conformation when interacting with ribosomal complexes.T he synthesis of proteins in prokaryotes is divided into three distinct processes: initiation, elongation, and termination. The initiation of translation in prokaryotes is directed by three initiation factors (IF1, IF2, and IF3) that govern the binding and positioning of the mRNA, as well as the initiator tRNA, and the joining of ribosomal subunits to form a 70S complex that is ready for the elongation stage of protein synthesis.IF2 is a GTPase that functions to position the initiator tRNA within the 30S ribosomal initiation complex (30S IC) and promotes its joining with the 50S ribosomal subunit to form a 70S ribosome. IF2 is encoded by a single copy of the infB gene and is completely conserved in bacteria (1). The flexible structure of the N terminus has the largest sequence variability among different species (2). Variability also exists within a species; for instance, Escherichia coli IF2 has three isoforms, which vary in the length of their N-terminal domain due to three distinct start sites for its translation initiation (1). The C-terminal part of IF2 (G, II, C1, and C2 domains) contains the highly conserved GTPase domain (G domain) and C2 domain, which interact with the initiator tRNA.The C2 domain recognizes and protects the formylated Met of the initiator tRNA from hydrolysis (3, 4). The formylation of Met results in a fivefold increase in the binding affinity of the tRNA and is made in a G-nucleotide-independent fashion (3, 5). This interaction permits IF2 to assist in positioning the initiator tRNA within a 30S initiation complex and guide the formation of a functional 30S IC on the establishment of the P-site codonanticodon interaction (2, 6, 7). A functional 30S IC is competent for the 50S ribosomal subunit to join, which is mediated by the formation of the intersubunit salt bridges via an interaction between IF2 and L12 (8). Once the 50S ribosomal subunit joins, IF2 comes into contact with the GTPase activation c...

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Section: Discussionmentioning

confidence: 68%

“…No structure from full-length IF2 at atomic resolution has previously been determined. Until recently, the only structural information at atomic resolution on IF2 has been NMR models of four separate domains of IF2 (N, G, C1, and C2) (12)(13)(14)(15).…”

mentioning

confidence: 99%

Initiation factor 2 crystal structure reveals a different domain organization from eukaryotic initiation factor 5B and mechanism among translational GTPases

Eiler

Lin

Simonetti

et al. 2013

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

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“…Furthermore, it is clear from the cryo-EM structure that domains C1 and C2 form a continuous, elongated bipartite structure without a long helix connecting them, in contrast to what was observed in the isolated a/eIF5B (20). Consistently, the C1 and C2 domains are rather flexible elements in the isolated factor, as illustrated by their variable conformations (12,21). In conclusion, S12 could be the stabilizing element sought for, perhaps through a small point contact at the end of helix 8 (Fig.…”

Section: Cryo-em Structures Of 30s and 70smentioning

confidence: 78%

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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“…Allosteric effects are usually mediated via structural rearrangements; however, allostery mediated by changes in the conformational entropy without alterations of the overall structure is well documented5152. Recent investigations of bacterial initiation factor IF2 showed profound structural rearrangements in the GDP-bound as compared to the apo-state95354, in agreement with complementary biochemical experiments demonstrating the functional differences between the two8. However, in the case of EF-G, an absence of significant structural rearrangements in EF-G in the presence of G-nucleotides was suggested on the basis of Small Angle X-ray Scattering (SAXS)55 and analysis of the changes in the Solvent Accessible Area (SAA) inferred from the ITC data19.…”

Section: Discussionmentioning

confidence: 99%

GTPases IF2 and EF-G bind GDP and the SRL RNA in a mutually exclusive manner

Mitkevich

Shyp

Petrushanko

et al. 2012

Sci Rep

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Translational GTPases (trGTPases) are involved in all four stages of protein biosynthesis: initiation, elongation, termination and ribosome recycling. The trGTPases Initiation Factor 2 (IF2) and Elongation Factor G (EF-G) respectively orchestrate initiation complex formation and translocation of the peptidyl-tRNA:mRNA complex through the bacterial ribosome. The ribosome regulates the GTPase cycle and efficiently discriminates between the GDP- and GTP-bound forms of these proteins. Using Isothermal Titration Calorimetry, we have investigated interactions of IF2 and EF-G with the sarcin-ricin loop of the 23S rRNA, a crucial element of the GTPase-associated center of the ribosome. We show that binding of IF2 and EF-G to a 27 nucleotide RNA fragment mimicking the sarcin-ricin loop is mutually exclusive with that of GDP, but not of GTP, providing a mechanism for destabilization of the ribosome-bound GDP forms of translational GTPases.

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Structural Dynamics of Bacterial Translation Initiation Factor IF2

Cited by 27 publications

References 66 publications

Initiation factor 2 crystal structure reveals a different domain organization from eukaryotic initiation factor 5B and mechanism among translational GTPases

Initiation factor 2 crystal structure reveals a different domain organization from eukaryotic initiation factor 5B and mechanism among translational GTPases

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

GTPases IF2 and EF-G bind GDP and the SRL RNA in a mutually exclusive manner

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