Solution Structure of the Ribosome-binding Domain ofE. coliTranslation Initiation Factor IF3. Homology with the U1A Protein of the Eukaryotic Spliceosome

Garcı́a, Carlos; Fortier, Pierre-Louis; Blanquet, Sylvain; Lallemand, Jean‐Yves; Dardel, Frédéric

doi:10.1006/jmbi.1995.0615

Cited by 68 publications

(55 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…6, panel v); only the host-encoded IF3 (deleted for 55 aa from the N terminus) is found in the 30S fraction. Therefore, our observations agree with the earlier in vitro data (7,27) and indicate that in vivo binding of the NTD to the ribosome must be poor or transient.…”

Section: Functions Of the Domains Of Initiation Factorsupporting

confidence: 92%

“…Their conclusions, which were gleaned from in vitro experiments, were also supported by nuclear magnetic resonance (NMR) spectroscopy (27,28), which in turn showed that most of the IF3 residues interacting with the ribosome were present in the CTD. Every IF3 mutation which led to ribosome-binding defects was mapped to the CTD, while in vivo studies performed with point mutants of IF3 have consistently indicated the role of residues of the NTD in the fidelity function of IF3 (5,11,12,29,30).…”

Section: Discussionmentioning

confidence: 85%

See 1 more Smart Citation

Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits

2017

View full text Add to dashboard Cite

Initiation factor 3 (IF3) is one of the three conserved prokaryotic translation initiation factors essential for protein synthesis and cellular survival. Bacterial IF3 is composed of a conserved architecture of globular N-and C-terminal domains (NTD and CTD) joined by a linker region. IF3 is a ribosome antiassociation factor which also modulates selection of start codon and initiator tRNA. All the functions of IF3 have been attributed to its CTD by in vitro studies. However, the in vivo relevance of these findings has not been investigated. By generating complete and partial IF3 (infC) knockouts in Escherichia coli and by complementation analyses using various deletion constructs, we show that while the CTD is essential for E. coli survival, the NTD is not. Polysome profiles reaffirm that CTD alone can bind to the 30S ribosomal subunit and carry out the ribosome antiassociation function. Importantly, in the absence of the NTD, bacterial growth is compromised, indicating a role for the NTD in the fitness of cellular growth. Using reporter assays for in vivo initiation, we show that the NTD plays a crucial role in the fidelity function of IF3 by avoiding (i) initiation from non-AUG codons and (ii) initiation by initiator tRNAs lacking the three highly conserved consecutive GC pairs (in the anticodon stem) known to function in concert with IF3. IMPORTANCE Initiation factor 3 regulates the fidelity of eubacterial translation initiation by ensuring the formation of an initiation complex with an mRNA bearing a canonical start codon and with an initiator tRNA at the ribosomal P site. Additionally, IF3 prevents premature association of the 50S ribosomal subunit with the 30S preinitiation complex. The significance of our work in Escherichia coli is in demonstrating that while the C-terminal domain alone sustains E. coli for its growth, the N-terminal domain adds to the fidelity of initiation of protein synthesis and to the fitness of the bacterial growth.KEYWORDS initiation with AUA, initiation with AUU, 3GC base pairs, initiator tRNA T he process of translation initiation is the most highly regulated step of protein synthesis, where the three initiation factors serve to establish this tight scrutiny. Initiation factor 3 (IF3) is one such factor which acts as an antiassociation factor for the two ribosomal subunits (1). IF3 from Escherichia coli is composed of 180 amino acids (aa) and is encoded by the essential infC gene (2). Structurally, IF3 can be divided into globular N-and C-terminal domains (NTD and CTD) joined by a linker region. The most important functions of IF3 include ribosome antiassociation (1, 3), shifting 30S-bound mRNA from standby to the P site (4), and its fidelity functions (5, 6). The fidelity function of IF3 entails ejection of incorrect (elongator) tRNAs to allow preferential selection of

show abstract

Section: Functions Of the Domains Of Initiation Factorsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 85%

Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits

2017

View full text Add to dashboard Cite

show abstract

“…As crystals containing the entire IF3 molecule or IF3N could not be produced owing to tight crystal contacts, IF3N and IF3 linker regions were docked onto the T30S complexed with IF3C with its known crystal structures (9), taking into account the flexibility of the linker as indicated by NMR studies (19,20). In its docked location, IF3N is positioned close to the P-site (Figure 1a), leaving sufficient space for cognate tRNA binding, but is too tight for allowing nearcognate or noncognate interactions (48), indicating that IF3N discriminates against noncognate tRNA by space exclusion principles.…”

Section: Initiation Of the Translation Process Initiation Factor 3: Amentioning

confidence: 99%

Ribosomal Crystallography: Initiation, Peptide Bond Formation, and Amino Acid Polymerization are Hampered by Antibiotics

Yonath

Bashan

2004

Annu. Rev. Microbiol.

View full text Add to dashboard Cite

■ Abstract High-resolution structures of ribosomal complexes revealed that minute amounts of clinically relevant antibiotics hamper protein biosynthesis by limiting ribosomal mobility or perturbing its elaborate architecture, designed for navigating and controlling peptide bond formation and continuous amino acid polymerization. To accomplish this, the ribosome contributes positional rather than chemical catalysis, provides remote interactions governing accurate substrate alignment within the flexible peptidyl-transferase center (PTC) pocket, and ensures nascent-protein chirality through spatial limitations. Peptide bond formation is concurrent with aminoacylatedtRNA 3 end translocation and is performed by a rotatory motion around the axis of a sizable ribosomal symmetry-related region, which is located around the PTC in all known crystal structures. Guided by ribosomal-RNA scaffold along an exact pattern, the rotatory motion results in stereochemistry that is optimal for peptide bond formation and for nascent protein entrance into the exit tunnel, the main target of antibiotics targeting ribosomes. By connecting the PTC, the decoding center, and the tRNA entrance and exit regions, the symmetry-related region can transfer intraribosomal signals, guaranteeing smooth processivity of amino acid polymerization. CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . INTRODUCTIONRibosomes are the universal cellular riboprotein assemblies that catalyze amino acid polymerization into proteins. In this process, called protein biosynthesis, the ribosomes interact with the cellular environment, select the genetic frame to be translated, form the peptide bonds, and act as polymerases to ensure smooth and efficient elongation of the newly born protein chains. The ribosomes are composed of two riboprotein subunits of unequal size that associate upon the initiation of the process and dissociate at its termination. Protein biosynthesis is performed by a cooperative effort of both subunits, and the ribosome possesses features that facilitate transmission between its various functional sites, leading to efficient processivity of protein formation essential for cell vitality. The small ribosomal subunit facilitates the initiation of the translation process and is involved in selecting the frame to be translated, decoding the genetic message, and controlling the fidelity of codon-anticodon interactions. The large ribosomal subunit forms the peptide bond, ensures smooth amino acid polymerization, and channels the nascent proteins through their exit tunnel. Messenger RNA (mRNA) carries the genetic instructions to the ribosome, and aminoacylated transfer RNA (tRNA) molecules deliver the amino acids within a ternary complex containing GTP and the elongation factor Tu. The ribosome possesses three tRNA binding sites. The A-site hosts the aminoacylated-tRNA, the P-site hosts the peptidyl tRNA, and the E-site designates the location of the exiting free tRNA once a p...

show abstract

“…It consists of C and N terminus domains (IF3C and IF3N) connected by a rather long lysine-rich linker region. The structure of the entire protein has not been determined, but NMR (22,23) and X-ray structures of the N-and C-terminal domains have been reported (8,36). The interdomain linker appears as a rigid alpha-helix only in the crystals containing it YONATH and IF3N.…”

Section: Conformational Mobility: the Key For Subunit Association DImentioning

confidence: 99%

The Search and Its Outcome: High-Resolution Structures of Ribosomal Particles from Mesophilic, Thermophilic, and Halophilic Bacteria at Various Functional States

Yonath

2002

Annu. Rev. Biophys. Biomol. Struct.

View full text Add to dashboard Cite

We determined the high-resolution structures of large and small ribosomal subunits from mesophilic and thermophilic bacteria and compared them with those of the thermophilic ribosome and the halophilic large subunit. We confirmed that the elements involved in intersubunit contacts and in substrate binding are inherently flexible and that a common ribosomal strategy is to utilize this conformational variability for optimizing its functional efficiency and minimizing nonproductive interactions. Under close-to-physiological conditions, these elements maintain well-ordered characteristic conformations. In unbound subunits, the features creating intersubunit bridges within associated ribosomes lie on the interface surface, and the features that bind factors and substrates reach toward the binding site only when conditions are ripe. 257 Annu. Rev. Biophys. Biomol. Struct. 2002.31:257-273. Downloaded from www.annualreviews.org Access provided by 54.191.190.102 on 05/12/18. For personal use only. 258 YONATH INTRODUCTIONRibosomes are the universal cellular organelles that catalyze the sequential polymerization of amino acids according to the genetic blueprint encoded in the mRNA. They are built of two subunits that associate for performing this task. The larger subunit creates the peptide bonds and provides the path for the progression of the nascent proteins. The smaller subunit has key roles in the initiation of the process, in decoding the genetic message, in discriminating against non-and near-cognate aminoacylated tRNA molecules, in controlling the fidelity of codon-anti-codon interactions, and in mRNA/tRNA translocation. The prokaryotic large ribosomal subunit (50S) has a molecular weight of 1.5 × 10 6 Dalton and contains two RNA chains with a total of ∼3000 nucleotides and ∼35 proteins. The small ribosomal subunit (called 30S) has a molecular weight of 8.5 × 10 5 Dalton and contains one RNA chain of over 1500 nucleotides and ∼20 proteins.Over two decades ago, we initialized a long and demanding search for the determination of the three-dimensional structure of the ribosome by X-ray crystallography (74). The key to high-resolution data was to crystallize homogenous preparations under conditions similar to their in situ environments or to induce a selected conformation after the crystals were formed. Relatively robust ribosomal particles were chosen, assuming that they would deteriorate less during preparation and therefore provide more homogenous starting materials for crystallization.The first crystals to yield some crystallographic information (e.g., symmetry, unit cell parameters, and resolution) were of the large subunit from Bacillus stearothermophilus (71) and Haloarcula marismortui (H50S) (39,66). Shortly afterward, we characterized crystals of the small subunit from Thermus thermophilus (T30S) (72). Microcrystals of the same source were grown independently at approximately the same time (64).An alternative approach was to design complexes containing ribosomes at defined functional stages, such as o...

show abstract

Solution Structure of the Ribosome-binding Domain ofE. coliTranslation Initiation Factor IF3. Homology with the U1A Protein of the Eukaryotic Spliceosome

Cited by 68 publications

References 0 publications

Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits

Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits

Ribosomal Crystallography: Initiation, Peptide Bond Formation, and Amino Acid Polymerization are Hampered by Antibiotics

The Search and Its Outcome: High-Resolution Structures of Ribosomal Particles from Mesophilic, Thermophilic, and Halophilic Bacteria at Various Functional States

Contact Info

Product

Resources

About