Translation Initiation Factor 3 Regulates Switching between Different Modes of Ribosomal Subunit Joining

doi:10.1016/j.jmb.2014.09.024

Journal of Molecular Biology

Volume 427, Issue 9, 8 May 2015, Pages 1801-1818

https://doi.org/10.1016/j.jmb.2014.09.024 Get rights and content

Highlights

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Joining of the 50S subunit to the 30S IC during bacterial translation initiation is regulated by three protein IFs.
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An smFRET assay was developed to probe interactions between IF2 and the GAC of the 50S subunit during subunit joining.
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30S IC-bound IF3 imparts reversibility to IF2-mediated subunit joining and influences interactions between IF2 and the GAC.
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Discrete modes of subunit joining were identified that resulted in 70S ICs with different lifetimes and conformations.
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IF3 modulates the intersubunit contacts formed during subunit joining, likely by regulating the conformation of the 30S IC.

Abstract

Ribosomal subunit joining is a key checkpoint in the bacterial translation initiation pathway during which initiation factors (IFs) regulate association of the 30S initiation complex (IC) with the 50S subunit to control formation of a 70S IC that can enter into the elongation stage of protein synthesis. The GTP-bound form of IF2 accelerates subunit joining, whereas IF3 antagonizes subunit joining and plays a prominent role in maintaining translation initiation fidelity. The molecular mechanisms through which IF2 and IF3 collaborate to regulate the efficiency of 70S IC formation, including how they affect the dynamics of subunit joining, remain poorly defined. Here, we use single-molecule fluorescence resonance energy transfer to monitor the interactions between IF2 and the GTPase-associated center (GAC) of the 50S subunit during real-time subunit joining reactions in the absence and presence of IF3. In the presence of IF3, IF2-mediated subunit joining becomes reversible, and subunit joining events cluster into two distinct classes corresponding to formation of shorter- and longer-lifetime 70S ICs. Inclusion of IF3 within the 30S IC was also found to alter the conformation of IF2 relative to the GAC, suggesting that IF3's regulatory effects may stem in part from allosteric modulation of IF2–GAC interactions. The results are consistent with a model in which IF3 can exert control over the efficiency of subunit joining by modulating the conformation of the 30S IC, which in turn influences the formation of stabilizing intersubunit contacts and thus the reaction's degree of reversibility.

Graphical abstract

Introduction

Translation initiation is a multistep process in which a functional ribosomal complex is assembled at the start codon of a messenger RNA (mRNA) template in preparation for a new round of protein synthesis. The canonical initiation pathway in bacteria begins with binding of initiation factors (IFs) to the small, 30S, ribosomal subunit; recruitment of the mRNA and initiator formyl-methionyl transfer RNA (fMet-tRNA^fMet); and formation of codon–anticodon base pairing between the tRNA anticodon and mRNA start codon. The resulting intermediate, known as the 30S initiation complex (IC), can subsequently associate with the large, 50S subunit to form a 70S IC that is competent to begin peptide chain elongation [1], [2]. The subunit joining step constitutes an important regulatory checkpoint during initiation, which may be up-regulated or down-regulated in order to accelerate 70S IC formation from correctly assembled 30S ICs, to prevent formation of aberrant 70S ICs containing non-initiator tRNA or incorrect start codons, and to fine-tune the efficiency of translation initiation on different mRNA templates [3], [4], [5], [6].

The three canonical IFs, IF1, IF2, and IF3, play prominent roles in regulating the rate and fidelity of individual steps of translation initiation, including 30S IC assembly and subunit joining. Previous ensemble biochemical and biophysical studies have shown that the universally conserved GTPase IF2 facilitates recruitment and selection of fMet-tRNA^fMet on the 30S IC during 30S IC assembly [7], [8] and subsequently accelerates subunit joining [3], [4], [9]. These studies have also shown that IF3 has a proofreading function during initiation, discriminating against non-canonical mRNA start codons and non-initiator tRNAs in favor of correct codon–anticodon pairing between a canonical mRNA start codon and fMet-tRNA^fMet during both 30S IC assembly [7], [10], [11] and subunit joining [5], [6]. During subunit joining, IF3 counteracts the stimulatory effect of IF2 by inhibiting the reaction to varying extents depending on the composition of the 30S IC [3], [4], [5], [12]. Finally, these studies have shown that IF1 enhances the respective activities of IF2 and IF3 during 30S IC assembly and subunit joining [3], [4], [13], [14]. The opposing effects of IF2 and IF3 on the rate of subunit joining are likely important in appropriately balancing the dual requirements of speed and accuracy during initiation [4] and may permit modulation of subunit joining efficiencies in a context-specific manner.

The stimulatory effect of IF2 on subunit joining can be attributed to the formation of favorable interactions with the intersubunit surface of the core of the 50S subunit and with components within the GTPase-associated center (GAC) of the 50S subunit [15], [16], [17], [18], which consists of 23S ribosomal RNA helices 42–44 and ribosomal proteins L10, L11, and L7/L12. Inhibition by IF3, on the other hand, is thought to be a consequence of its binding to the “platform” domain of the 30S subunit, where it can sterically block the formation of key intersubunit interactions, termed intersubunit bridges, at the interface between the 30S and 50S subunits [19], [20].

Different models have been proposed for how IF3 exerts control over subunit joining. One model posits that IF3 must dissociate from the 30S IC prior to successful subunit joining and that the rate of subunit joining is dictated by the rate of IF3 dissociation [4]. An alternate model proposes that IF1- and IF3-dependent conformational changes of the 30S IC control whether subunit joining can result in the formation of a thermodynamically stable 70S IC and that IF3 dissociation occurs subsequent to subunit joining [5]. IF3 is a structurally dynamic protein consisting of two globular domains connected by a flexible linker, which has recently been shown to adopt multiple interdomain conformations on the 30S IC that may be coupled to global conformational changes of the entire 30S IC [21]. These results lend support to the possibility that the 30S IC can undergo conformational changes that modulate the accessibility of intersubunit bridges and hence the rate of subunit joining, without requiring IF3 to dissociate. They also raise the question of whether and to what extent IF3 influences the interactions of other 30S IC components, such as fMet-tRNA^fMet and IF2, with the incoming 50S subunit. Addressing this question would furnish a more complete understanding of the mechanism through which IF3 regulates subunit joining.

Previous studies have demonstrated the efficacy of single-molecule fluorescence methods in characterizing conformational and temporal dynamics of the ribosome and its ligands during translation initiation [21], [22], [23], [24]. Here, we report the use of a single-molecule fluorescence resonance energy transfer (smFRET) approach to specifically characterize the effect of IF3 on IF2-mediated subunit joining. Direct observation of individual subunit joining events has the potential to uncover structural and kinetic features underlying regulation by IF3 that have been hidden or obscured in previous ensemble-averaged measurements. Subunit joining reactions were monitored in real time based on energy transfer between fluorescence resonance energy transfer (FRET)-donor-labeled IF2 on the 30S IC and FRET-acceptor-labeled ribosomal protein L11 within the GAC. This strategy allows detection of conformational rearrangements between IF2 and the GAC during subunit joining, which have been suggested to play a functional role in this process [25], [26], and permits an assessment of the extent to which the presence of IF3 on the 30S IC alters the interactions that IF2 forms with the 50S subunit.

We find that the presence of IF3 dramatically alters the dynamics of subunit joining. In the absence of IF3, subunit joining results in the formation of a highly stable, long-lifetime 70S IC. When IF3 is present within the 30S IC, however, subunit joining becomes reversible and two distinct types of subunit joining events are observed, corresponding to formation of 70S ICs with short and intermediate lifetimes and distinct distributions of relative IF2–GAC conformations. These results suggest that IF3 can exert control over subunit joining by modulating the conformation of the 30S IC and that the IF3-bound 30S IC can transition between at least two conformations that interact more weakly or more strongly with the 50S subunit. The relative occurrence of short- and intermediate-lifetime 70S ICs was found to be modulated by changes in the solution concentration of IF3 and by the use of a loss-of-function IF3 point mutant [27], [28], suggesting that the equilibrium between the two 30S IC conformations can be regulated and that this could provide a mechanism to control the efficiency and fidelity of translation initiation. Thus, IF3-dependent modulation of the energetics of subunit joining may allow fine-tuning of the rate of 70S IC formation and entry into elongation within different cellular contexts and in response to different cellular cues.

Section snippets

Development of an IF2–L11 smFRET signal to monitor subunit joining

We devised a single-molecule approach to monitor the interactions between 30S IC-bound IF2 and L11 within the GAC during real-time subunit joining reactions (Fig. 1). An IF2(S672C) point mutant was constructed and site-specifically labeled with Cy3 FRET donor fluorophore to generate (Cy3)IF2. Escherichia coli contains three naturally occurring isoforms of IF2 (α, β, and γ) that differ in length of the N-terminus but that are each fully capable of promoting translation initiation in vitro [29];

Discussion

Our smFRET studies have revealed the existence of multiple, discrete modes of subunit joining during translation initiation. Joining of the 50S subunit to 30S IC_−IF3 leads to the formation of a long-lifetime 70S IC, whereas joining of the 50S subunit to 30S IC_+IF3 can result in the formation of short- or intermediate-lifetime 70S ICs (Fig. 7). Both short- and intermediate-lifetime 70S ICs were observed to undergo dissociation into free subunits, providing direct evidence that IF2-mediated

Purification and fluorescent labeling of translation components

The IF2 construct used for smFRET experiments was generated by first introducing the S672C point mutation into a cloned copy of the γ-isoform of E. coli IF2 using the QuikChange II-E Site-Directed Mutagenesis Kit (Stratagene). Six-histidine-tagged IF2 S672C was purified by Ni-NTA chromatography, followed by cleavage of the affinity tag and purification by cation-exchange chromatography [56]. Fluorescent labeling was achieved by reaction with 10-fold molar excess of Cy3-maleimide followed by

Acknowledgments

This work was supported by grants from the Burroughs Wellcome Fund (CABS 1004856) and the US National Institutes of Health (R01 GM 084288) to R.L.G., as well as a Camille Dreyfus Teacher-Scholar Award to R.L.G. We thank Prof. Walter Hill for the kind gift of wild-type and L11-deletion strains used to purify ribosomes, as well as Prof. Bruno Klaholz for providing atomic coordinates of cryo-EM reconstructions of the 70S IC. We are grateful to members of the Gonzalez laboratory and the laboratory

References (61)

A. Antoun et al.
How initiation factors maximize the accuracy of tRNA selection in initiation of bacterial protein synthesis
Mol Cell
(2006)
P. Milon et al.
Kinetic checkpoint at a late step in translation initiation
Mol Cell
(2008)
C. Grigoriadou et al.
The translational fidelity function of IF3 during transition from the 30 S initiation complex to the 70 S initiation complex
J Mol Biol
(2007)
C. Grigoriadou et al.
A quantitative kinetic scheme for 70 S translation initiation complex formation
J Mol Biol
(2007)
G.S. Allen et al.
The cryo-EM structure of a translation initiation complex from Escherichia coli
Cell
(2005)
C. Huang et al.
The ribosomal stalk plays a key role in IF2-mediated association of the ribosomal subunits
J Mol Biol
(2010)
A. Dallas et al.
Interaction of translation initiation factor 3 with the 30S ribosomal subunit
Mol Cell
(2001)
R.A. Marshall et al.
GTP hydrolysis by IF2 guides progression of the ribosome into elongation
Mol Cell
(2009)
L. Brandi et al.
The translation initiation functions of IF2: targets for thiostrepton inhibition
J Mol Biol
(2004)
D. Maar et al.
A single mutation in the IF3 N-terminal domain perturbs the fidelity of translation initiation at three levels
J Mol Biol
(2008)

N.R. Nyengaard et al.

Tandem translation of E. coli initiation factor IF2 beta: purification and characterization in vitro of two active forms

Biochem Biophys Res Commun

(1991)

A. Roll-Mecak et al.

X-Ray structures of the universal translation initiation factor IF2/eIF5B: conformational changes on GDP and GTP binding

Cell

(2000)

N. Van Dyke et al.

Limitation of ribosomal protein L11 availability in vivo affects translation termination

J Mol Biol

(2002)

W.S. Bowen et al.

Interaction of thiostrepton and elongation factor-G with the ribosomal protein L11-binding domain

J Biol Chem

(2005)

J.B. Chaires et al.

The effect of initiation factor IF-3 on Escherichia coli ribosomal subunit association kinetics

J Biol Chem

(1981)

T. Ha

Single-molecule approaches embrace molecular cohorts

Cell

(2013)

D.N. Ermolenko et al.

Observation of intersubunit movement of the ribosome in solution using FRET

J Mol Biol

(2007)

U. Rawat et al.

Interactions of the release factor RF1 with the ribosome as revealed by cryo-EM

J Mol Biol

(2006)

J.M. Kavran et al.

Structure of the base of the L7/L12 stalk of the Haloarcula marismortui large ribosomal subunit: analysis of L11 movements

J Mol Biol

(2007)

J.S. Butler et al.

Escherichia coli protein synthesis initiation factor IF3 controls its own gene expression at the translational level in vivo

J Mol Biol

(1986)

R. Karimi et al.

Novel roles for classical factors at the interface between translation termination and initiation

Mol Cell

(1999)

J. Fei et al.

A highly purified, fluorescently labeled in vitro translation system for single-molecule studies of protein synthesis

Methods Enzymol

(2010)

J. Fei et al.

Coupling of ribosomal L1 stalk and tRNA dynamics during translation elongation

Mol Cell

(2008)

J.E. Bronson et al.

Learning rates and states from biophysical time series: a Bayesian approach to model selection and single-molecule FRET data

Biophys J

(2009)

B.S. Laursen et al.

Initiation of protein synthesis in bacteria

Microbiol Mol Biol Rev

(2005)

A. Simonetti et al.

A structural view of translation initiation in bacteria

Cell Mol Life Sci

(2009)

A. Antoun et al.

How initiation factors tune the rate of initiation of protein synthesis in bacteria

EMBO J

(2006)

D. Hartz et al.

Selection of the initiator tRNA by Escherichia coli initiation factors

Genes Dev

(1989)

P. Milon et al.

The ribosome-bound initiation factor 2 recruits initiator tRNA to the 30S initiation complex

EMBO Rep

(2010)

D. Hartz et al.

Domains of initiator tRNA and initiation codon crucial for initiator tRNA selection by Escherichia coli IF3

Genes Dev

(1990)

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Whereas the simultaneous binding of GTP and fMet-tRNAfMet confers an “active” conformation of IF2 which promotes rapid subunit joining (Antoun et al., 2003; Grunberg-Manago et al., 1975; Pavlov et al., 2011; Wang et al., 2015; Zorzet et al., 2010), IF3 induces an anti-association conformation of the 30S subunit, which is enhanced or alleviated depending on the mRNA sequence and correct start codon-anticodon interaction (Antoun et al., 2006b; Grigoriadou et al., 2007b; Milón et al., 2008). The antagonistic interplay between IF2 and IF3 fine-tunes the initiator tRNA selection and subunit joining, maintaining the balance between the speed and accuracy of initiation (Antoun et al., 2006b; MacDougall and Gonzalez, 2015). The maturation of the 30S IC to an elongation-ready 70S IC involves a 50S subunit binding step, a chemical GTP hydrolysis step and several factor dissociation events that occur on the millisecond to second timescale.
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In bacterial translational initiation, three initiation factors (IFs 1–3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1–3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNA^fMet) into the P site for start codon recognition.
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IF3 binds the 30S platform and is predicted to occlude the formation of central bridges B2b and B2a/d [81,83,84]. Functional studies show that both IF1 and IF3 negatively regulate subunit joining and enhance the fidelity of start codon selection [85–93]. IF2 is a GTPase that interacts with the 30S shoulder domain and the acceptor end of fMet-tRNA, extending more than 100 Å across the interface side of the 30S subunit [21,84].
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