A Quantitative Kinetic Scheme for 70 S Translation Initiation Complex Formation

Grigoriadou, Christina; Marzi, Stefano; Kirillov, Stanislas; Gualerzi, Claudio O.; Cooperman, Barry S.

doi:10.1016/j.jmb.2007.07.032

Cited by 87 publications

(214 citation statements)

References 36 publications

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“…S1, and Table 1). Biphasic increases in LS have been reported in earlier studies and were attributed to either two-step 50S docking (14) or functional heterogeneity of the 30SIC, in which a fraction of the 30S complexes exist in a dockingincompetent state at the time of mixing (16,24). The latter may be more pertinent to the current study, as our data most closely resemble those of Rodnina and coworkers (16).…”

Section: H69supporting

confidence: 87%

“…50S docking stimulates the GTPase activity of IF2 (13), presumably through the interaction between the sarcin-ricin loop and IF2 G domain, and triggers a large conformational change that favors factor dissociation (12). This is accompanied by movement of the initiator tRNA into the P/P site (14). IF3 inhibits subunit joining, an effect more pronounced in the presence of noncanonical codonanticodon base pairing (5,15).…”

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confidence: 99%

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Roles of helix H69 of 23S rRNA in translation initiation

Liu

Fredrick

2015

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

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Initiation of translation involves the assembly of a ribosome complex with initiator tRNA bound to the peptidyl site and paired to the start codon of the mRNA. In bacteria, this process is kinetically controlled by three initiation factors-IF1, IF2, and IF3. Here, we show that deletion of helix H69 (ΔH69) of 23S rRNA allows rapid 50S docking without concomitant IF3 release and virtually eliminates the dependence of subunit joining on start codon identity. Despite this, overall accuracy of start codon selection, based on rates of formation of elongation-competent 70S ribosomes, is largely uncompromised in the absence of H69. Thus, the fidelity function of IF3 stems primarily from its interplay with initiator tRNA rather than its anti-subunit association activity. While retaining fidelity, ΔH69 ribosomes exhibit much slower rates of overall initiation, due to the delay in IF3 release and impedance of an IF3-independent step, presumably initiator tRNA positioning. These findings clarify the roles of H69 and IF3 in the mechanism of translation initiation and explain the dominant lethal phenotype of the ΔH69 mutation.T ranslation initiation can be divided into two major stages in bacteria. The first stage involves assembly of the 30S initiation complex (30SIC). Facilitated by three initiation factors, initiator tRNA (N-formyl-methionyl-tRNA fMet , or fMet-tRNA fMet ) binds to the peptidyl (P) site of the 30S subunit and pairs with the start codon on the mRNA. During the second stage, the 50S subunit associates with the 30SIC and triggers dissociation of the initiation factors, leaving the 70S initiation complex (70SIC) with fMet-tRNA fMet in the P site, ready for elongation. Because initiation is the rate-limiting step of translation and establishes the reading frame, efficient and accurate assembly of the 70SIC is critical for cell survival.30SIC assembly can be considered a largely random-order process, although there is a preferred kinetic pathway of ligand binding. IF2 and IF3 are generally first to bind to the 30S subunit, followed by IF1 and fMet-tRNA fMet , whereas the timing of mRNA binding depends on its sequence context and cellular concentration (1). The three initiation factors reciprocally stabilize one another in the 30SIC, and their binding induces a conformational change of the subunit, including a clockwise rotation of the head domain (2). IF1 is an 8-kDa protein that binds to helix h44, the 530 loop, and the S12 region and blocks the 30S aminoacyl (A) site (3). IF2 is a multidomain ribosomedependent GTPase that makes extensive contacts with both the 30S subunit and fMet-tRNA fMet . Domains G3 and C1 of IF2 bind helix h5 and h14 of 16S rRNA, the N-terminal domain (NTD) interacts with S16 and IF1, and domain C2 recognizes the acceptor stem and fMet moiety of fMet-tRNA fMet (2, 4). These interactions contribute to functions of IF2 in increasing the on rate of fMet-tRNA fMet binding and discriminating against elongator tRNAs (5). IF3 consists of two globular domains connected by a flexible linker (6, 7)...

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Section: H69supporting

confidence: 87%

mentioning

confidence: 99%

Roles of helix H69 of 23S rRNA in translation initiation

Liu

Fredrick

2015

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

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“…There are conflicting reports on whether the release of inorganic phosphate from IF2 very rapidly follows GTP hydrolysis (38) or is slower by nearly one order of magnitude (16,17). In our subunit-joining smFRET experiments performed at the 100-ms time resolution, the semirotated intermediate of initiation was stabilized when GTP was replaced with GDPCP, whereas it was undetected in the majority of traces obtained in the presence of GTP.…”

Section: Discussionmentioning

confidence: 67%

“…GTP hydrolysis stimulated by the large ribosomal subunit triggers the release of IF2 (13, 14), whereas IF1 and IF3 likely disassociate from the ribosome concurrently (11) or shortly after subunit joining, but before the release of IF2 (12, 15). Although the specific functions of each initiation factor, as well as the order and kinetics of the different steps of initiation, have been extensively studied (12,(16)(17)(18)(19)(20), many molecular details of this process including the conformational rearrangements of the ribosome remain unclear.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 (21-24). However, these studies have not unambiguously determined which conformation is adopted by the ribosome on IF2-mediated subunit joining.…”

mentioning

confidence: 99%

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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“…1, left panel). 1,2,13,16,17 In eukaryotes, the small (40S) ribosomal subunit also serves as a central hub for initiation factors (i.e., eIF3, eIF1/1A/5 and the eIF2 GTP Met-tRNA i Met ternary complex (TC)) as well as mRNA and tRNAs. [12][13][14][15] After translation termination and ribosome recycling, the 40S and 60S subunits get dissociated, stripped of the mRNA, P-site deacylated tRNA and polypeptide release factor eRF1.…”

Section: Introductionmentioning

confidence: 99%

Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

Ghosh

Komar

2015

Translation

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Keywords: conserved ribosomal proteins, eukaryotic/yeast ribosome, eukaryote-specific extensions, eukaryotic translation initiation factors, protein synthesis, small ribosomal subunitHigh-resolution structures of yeast ribosomes have improved our understanding of the architecture and organization of eukaryotic rRNA and proteins, as well as eukaryote-specific extensions present in some conserved ribosomal proteins. Despite this progress, assignment of specific functions to individual proteins and/or eukaryotespecific protein extensions remains challenging. It has been suggested that eukaryote-specific extensions of conserved proteins from the small ribosomal subunit may facilitate eukaryote-specific reactions in the initiation phase of protein synthesis. This review summarizes emerging data describing the structural and functional significance of eukaryotespecific extensions of conserved small ribosomal subunit proteins, particularly their possible roles in recruitment and spatial organization of eukaryote-specific initiation factors.

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A Quantitative Kinetic Scheme for 70 S Translation Initiation Complex Formation

Cited by 87 publications

References 36 publications

Roles of helix H69 of 23S rRNA in translation initiation

Roles of helix H69 of 23S rRNA in translation initiation

Initiation factor 2 stabilizes the ribosome in a semirotated conformation

Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

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