Translation initiation factor IF3: two domains, five functions, one mechanism?

Petrelli, Dezemona; Teana, Anna La; Garofalo, Cristiana; Spurio, Roberto; Pon, Cynthia L.; Gualerzi, Claudio O.

doi:10.1093/emboj/20.16.4560

Cited by 108 publications

(139 citation statements)

References 56 publications

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“…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%

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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

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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

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Section: Functions Of the Domains Of Initiation Factorsupporting

confidence: 92%

“…Although, according to an earlier study (7), the CTD of IF3 is supposed to possess all the functions of the molecule, no study has been able to show if the CTD of IF3 can sustain a bacterial cell. In fact, overexpression of the individual CTD of IF3 has been suggested to be toxic (11).…”

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confidence: 99%

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

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“…The first function of IF3 is related to the accuracy of initiation, because IF3 increases the dissociation rate of noncanonical 30SIC (56), a function that is restricted to 30SIC rather than to 70SIC (17), leaving IF3's important role for 70S scanning as its main second function.…”

Section: Discussionmentioning

confidence: 99%

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Yamamoto

Wittek

Gupta

et al. 2016

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

106

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According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1-IF3). Here, we describe a novel type of initiation termed "70S-scanning initiation," where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine-Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.protein synthesis | ribosomal functions | translational initiation | 30S-binding initiation | 70S-scanning initiation I t is textbook knowledge that 30S subunits initiate protein synthesis in bacteria; they recognize the initiation site of the mRNA composed of the Shine-Dalgarno (SD) sequence, the AUG codon, and fMet-tRNA, together with three initiation factors (IFs) forming the 30S initiation complex (30SIC). Association of the large 50S subunit triggers the release of the IFs, leading to the 70S initiation complex (70SIC) that enters the elongation phase of translation (reviewed in 1). We term this initiation path the "30S-binding mode" of bacterial initiation. After elongation and termination, it is thought that the ribosome dissociates into its subunits, thus providing 30S subunits for the next round of initiation.The functional role of IF2 is well defined. It can bind directly to the 30S, providing a docking site for fMet-tRNA (2), but it can also enter the 30S subunit as ternary complex fMet-tRNA•IF2•GTP (3). Both IF2 and IF3 are essential for viability. IF3 has a binding site at the 30S interface (4), which explains its antiassociation effect (5, 6), as well as its role in dissociation of the terminating 70S ribosome (7). However, the in vivo concentration of IF3 is 100-fold less (8) than required for full dissociation of 70S in vitro (4). Evidence for the presence of IF3 on 70S ribosomes was reported (9), indicating that the functional spectrum of IF3 is possibly not restricted to an antiassociation effect. Both IF3 and IF2 are also responsible for the fidelity of decoding the initiation AUG by fMet-tRNA Met f at the P site of 30S subun...

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“…They treated the insoluble chromatin-containing fraction, obtained during chromatin fractionation, with DNase I and RNase A, respectively, and found that Sso10b was liberated in both cases. Furthermore, structural comparison had shown that Sac10b members shared their mixed / -fold with IF3-C, the C-terminal domain of translation initiation factor IF3 [27], which had the full biological activity of the entire protein molecule [55] and interacted with 16S rRNA and tRNA fMet in the 30S ribosomal subunit detected by a combination of hydroxyl radical footprinting and directed hydroxyl radical probing with Fe(II)-derivatized IF3 [56].…”

Section: Functional Divergence -Rna-binding Proteinsmentioning

confidence: 99%

The Archaeal Sac10b Protein Family: Conserved Proteins with Divergent Functions

Xuan¹,

Feng²

2012

CPPS

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Abstract:Here we review the present state of structural and functional studies of the Sac10b protein family, a class of highly conserved 10 kDa nucleic acid-binding proteins in archaea. Based on biochemical and structural studies, these proteins were originally assigned a role in the structural organization of chromatin; Sac10b proteins of hyperthermophilic archaea, for example, showed tight, unspecific DNA binding. More recently, however, Sac10b proteins of mesophilic archaea were found to interact preferentially with specific DNA sequences thereby affecting the expression of distinct genes. Furthermore, Sac10b proteins of hyperthermophilic, thermophilic and mesophilic archaea were also shown to bind to RNA with distinct affinities and specificities but functional consequences of RNA binding of these proteins, besides perhaps RNA stabilization, have not yet been observed. To better understand the physiological meaning of the various interactions of Sac10b proteins with nucleic acids, future work should concentrate on elucidating the molecular structures of complexes of Sac10b proteins of hyperthermophilic and mesophilic archaea with DNA and RNA. In addition, existing and new X-ray and NMR structures of individual hyperthermophilic Sac10b proteins may represent very good models for introducing thermostability especially in enzymes for industrial use.

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Translation initiation factor IF3: two domains, five functions, one mechanism?

Cited by 108 publications

References 56 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

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

The Archaeal Sac10b Protein Family: Conserved Proteins with Divergent Functions

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