Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome

Myasnikov, Alexander G.; Marzi, Stefano; Simonetti, Angelita; Giuliodori, Anna Maria; Gualerzi, Claudio O.; Yusupova, G.; Yusupov, Marat; Klaholz, Bruno P.

doi:10.1038/nsmb1012

Cited by 135 publications

(251 citation statements)

References 33 publications

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“…S7 E and F), suggesting that the formation of the semirotated conformation of the ribosome requires the presence of an aminoacylated initiator fMet-tRNA fMet . These results are consistent with cryo-EM reconstructions suggesting that the C-terminal domain of IF2 interacts with the acceptor end of initiator tRNA (21,22,29,30) and biochemical experiments showing that IF2 requires the formyl-methionyl moiety to catalyze efficient subunit joining (10). Noteworthy, virtually no rotated conformation corresponding to the 0.4 FRET state is observed in 70S ribosomes formed in the presence of fMet-tRNA fMet and IF2 (Fig.…”

Section: Joiningsupporting

confidence: 89%

“…Thus, neither IF1 nor IF3 contribute to the stabilization of the semirotated conformation of the 70S • IF2 IC. and induce intersubunit rotation in the 70S ribosome (21), prompting us to test whether IF2 • GDP can stabilize the semirotated conformation of the ribosome. However, neither in the absence of nucleotides (Fig.…”

Section: Joiningmentioning

confidence: 99%

“…Furthermore, in the Escherichia coli 70S • IF2 IC, the initiator tRNA was observed in an intermediate position between the classical P/P and hybrid P/E states that was named the P/I state (22). By contrast, the cryo-EM reconstruction of the Thermus thermophilus 70S • IF2 IC showed the ribosome in a conformation that was similar to the nonrotated state containing initiator tRNA bound in the classical P/P state (21). Moreover, the cryo-EM reconstruction of IF2 bound to the rotated, hybrid state ribosome has also been determined (23).…”

mentioning

confidence: 95%

“…However, these studies have not unambiguously determined which conformation is adopted by the ribosome on IF2-mediated subunit joining. Relatively low-resolution (>11 Å) cryo-EM reconstructions of a late intermediate of initiation, the 70S IC bound with IF2, suggested that the small ribosomal subunit in 70S • IF2 ICs is rotated ∼4-5°relative to the large ribosomal subunit, which is less than the degree of rotation observed in hybrid, fully rotated ribosomes (21)(22)(23). Furthermore, in the Escherichia coli 70S • IF2 IC, the initiator tRNA was observed in an intermediate position between the classical P/P and hybrid P/E states that was named the P/I state (22).…”

mentioning

confidence: 95%

“…Previous cryo-EM and FRET studies have suggested that rotation between ribosomal subunits may be involved in the transition from the initiation to the elongation phase of protein synthesis (21)(22)(23)(24). However, these studies have not unambiguously determined which conformation is adopted by the ribosome on IF2-mediated subunit joining.…”

mentioning

confidence: 99%

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Initiation factor 2 stabilizes the ribosome in a semirotated conformation

Ling

Ermolenko

2015

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

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Intersubunit rotation and movement of the L1 stalk, a mobile domain of the large ribosomal subunit, have been shown to accompany the elongation cycle of translation. The initiation phase of protein synthesis is crucial for translational control of gene expression; however, in contrast to elongation, little is known about the conformational rearrangements of the ribosome during initiation. Bacterial initiation factors (IFs) 1, 2, and 3 mediate the binding of initiator tRNA and mRNA to the small ribosomal subunit to form the initiation complex, which subsequently associates with the large subunit by a poorly understood mechanism. Here, we use single-molecule FRET to monitor intersubunit rotation and the inward/outward movement of the L1 stalk of the large ribosomal subunit during the subunit-joining step of translation initiation. We show that, on subunit association, the ribosome adopts a distinct conformation in which the ribosomal subunits are in a semirotated orientation and the L1 stalk is positioned in a half-closed state. The formation of the semirotated intermediate requires the presence of an aminoacylated initiator, fMet-tRNA fMet , and IF2 in the GTP-bound state. GTP hydrolysis by IF2 induces opening of the L1 stalk and the transition to the nonrotated conformation of the ribosome. Our results suggest that positioning subunits in a semirotated orientation facilitates subunit association and support a model in which L1 stalk movement is coupled to intersubunit rotation and/or IF2 binding.T he coordinated structural rearrangements of the ribosome and protein factors underlie the mechanism of translation. During the elongation phase of protein synthesis, the movement of tRNAs through the ribosome is accompanied by large-scale conformational changes, such as intersubunit rotation (1), the swiveling of the 30S subunit head (2), and the movement of a mobile domain of the large ribosomal subunit, the L1 stalk (3). Although the elongating ribosome likely samples a number of transient conformations, it predominantly adopts two main structural states: the nonrotated, classical state and the rotated, hybrid state (4). Translocation of tRNAs from the A and P to the P and E sites occurs through the formation of the intermediate hybrid A/P and P/E states, in which anticodon stem-loops of tRNAs are bound to the A and P site of the small subunit, whereas the acceptor ends are bound to the P and E sites of the large subunit, respectively (5). Hybrid state formation is coupled to a ∼7°-∼10°rotation of the body and platform of the small ribosomal subunit relative to the large ribosomal subunit and the inward movement of the 50S L1 stalk (6). Blocking intersubunit rotation by a covalent cross-link between subunits abolishes tRNA translocation (7). Furthermore, the antibiotics viomycin and neomycin inhibit tRNA translocation while trapping the ribosome in the rotated and semirotated conformations, respectively (8, 9). Hence, rearrangements of the ribosome are essential for translation elongation. However, the role of riboso...

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Section: Joiningsupporting

confidence: 89%

Section: Joiningmentioning

confidence: 99%

mentioning

confidence: 95%

mentioning

confidence: 95%

mentioning

confidence: 99%

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Initiation factor 2 stabilizes the ribosome in a semirotated conformation

Ling

Ermolenko

2015

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

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Protein Synthesis Initiation in Bacteria

Grunberg‐Manago¹,

Studer

Joseph

2007

Encyclopedia of Life Sciences

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Based in part on the previous version of this Encyclopedia of Life Sciences (ELS) article, Protein Synthesis Initiation in Bacteria by Marianne Grunberg-Manago.Translation initiation begins with the assembly of the pre-initiation complex followed by the formation of the initiation complex. The process of initiation then continues until the first peptide bond is formed; it is at this point that translation begins. The formation of the pre-initiation/initiation complex is often the rate-limiting step during the process of translation, as it is influenced by a number of translational regulatory mechanisms. As a result, the process of initiation can play a significant role in gene expression. Messenger RNA Selection -Shine and Dalgarno InteractionMessenger RNAs can easily be argued as one of the most diverse aspects of the translation initiation complex. These

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Protein Synthesis Initiation in Bacteria

Grunberg‐Manago

Studer

Joseph

2014

Encyclopedia of Life Sciences

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Ribosomes are responsible for translating the information in messenger ribonucleic acids (mRNAs) to synthesise proteins that the cell needs to carry out its function. Protein synthesis begins with the 30S ribosomal subunit recruiting a mRNA with the help of an initiator transfer RNA and three initiation factors. Successful recruitment of the mRNA results in the formation of the 30S pre‐initiation complex, which is followed by the joining of the 50S ribosomal subunit to form the 70S ribosome. Recent studies indicate the order in which the initiation factors bind and promote the steps in initiation complex formation. The formation of the pre‐initiation/initiation complex is often the rate‐limiting step during the process of translation, as it is influenced by a number of translational regulatory mechanisms. As a result, the process of initiation can play a significant role in gene expression. Key Concepts: Translation initiation is a key step for regulating gene expression. Initiation factors 1, 2 and 3 are essential proteins that promote the binding of the initiator tRNA to the 30S subunit P site and the joining of the 50S subunit. Initiation factors 1, 2 and 3 are critical for the positioning the mRNA in the correct reading frame for translation. Efficient requirement of mRNA by the 30S subunit depends on the Shine–Dalgarno sequence in the mRNA forming base pairs with the anti‐Shine–Dalgarno sequence located at the 3′‐end of 16S rRNA. Kinetic studies have shown the order in which initiation factors bind to the 30S subunit. Cryo‐EM and crystallographic work have revealed the three‐dimensional location of initiation factors on the 30S subunit and the 70S ribosome.

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Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome

Cited by 135 publications

References 33 publications

Initiation factor 2 stabilizes the ribosome in a semirotated conformation

Initiation factor 2 stabilizes the ribosome in a semirotated conformation

Protein Synthesis Initiation in Bacteria

Protein Synthesis Initiation in Bacteria

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