Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs

Tedin, Karsten; Moll, Isabella; Grill, Sonja; Resch, Armin; Graschopf, Anton; Gualerzi, Claudio O.; Bläsi, Udo

doi:10.1046/j.1365-2958.1999.01147.x

Cited by 81 publications

(98 citation statements)

References 47 publications

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“…Thus, in accordance with the toeprinting experiments shown in Figs. 1 and 3, and with our previous studies [4,6], enhanced levels of IF2 and IF3, respectively, increase and decrease translation events at 5P-terminal start codons in vivo. The presence of both plasmid borne initiation factor genes in strain MC4100FPVWH103(pAUGinfC; pLBYC8) resulted in a V5-fold increase when compared to the control strain.…”

Section: The In Vivo Translational E¤ciency Of a Leaderless Reportersupporting

confidence: 88%

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Modulation of ribosomal recruitment to 5′‐terminal start codons by translation initiation factors IF2 and IF3

et al. 2001

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Edited by Lev KisselevAbstract Sequence determinants and structural features of the RNA govern mRNA^ribosome interaction in bacteria. However, ribosomal recruitment to leaderless mRNAs, which start directly with the AUG start codon and do not bear a Shine^Dalgarno sequence like canonical mRNAs, does not appear to rely on 16S rRNA^mRNA interactions. Here, we have studied the effects of translation initiation factors IF2 and IF3 on 30S initiation at a 5P P-terminal AUG and at a competing downstream canonical ribosome binding site. We show that IF2 affects the forward kinetics of 30S initiation complex formation at the 5P P-terminal AUG as well as the stability of these complexes. Moreover, the IF2:IF3 molar ratio was found to play a decisive role in translation initiation of a leaderless mRNA both in vitro and in vivo indicating that the translational efficiency of an mRNA is not only intrinsically determined but can be altered depending on the availability of components of the translational machinery. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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Section: The In Vivo Translational E¤ciency Of a Leaderless Reportersupporting

confidence: 88%

“…Plasmid pAUGinfC is a derivative of the low copy number plasmid pK184 and encodes an E. coli infC allele equipped with an AUG start codon [4]. Plasmid pLBYC8 is a derivative of the high copy number plasmid pTrc99A (Pharmacia Biotech.)…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

Modulation of ribosomal recruitment to 5′‐terminal start codons by translation initiation factors IF2 and IF3

et al. 2001

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“…2A, Middle). Furthermore, IF3 was thought to inhibit initiation of 70S ribosomes due to its 70S-dissociation activity (20,36), whereas we find that IF3 is essential for lmRNA translation, although IF1 is not involved.…”

Section: Resultscontrasting

confidence: 66%

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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“…In particular, IF3 has been shown to play a significant role in start codon selection. 32,33,34 To examine the possibility that the hyperactive phenotype produced by mutations at 966 and 967 is due to an effect on IF3 binding, we cloned IF1, IF2 and IF3 into a pACYC177 35 derivative constructed in our lab, pKAN6, and placed them under control of the inducible araBAD promoter. 36, 37 As a negative control, we also measured the effect of initiation factor over-expression on GFP translation by ribosomes containing the G693U mutation in 16S rRNA.…”

Section: Mutations At M 2 G966 and M 5 C967 Produce Hyperactive Ribosmentioning

confidence: 99%

Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA

Saraiya¹,

Lamichhane

Chow

et al. 2008

Journal of Molecular Biology

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SummaryThe 970 loop (helix 31) of Escherichia coli 16S rRNA contains two modified nucleotides, m 2 G966 and m 5 C967. Positions A964, A969 and C970 are conserved among the Bacteria, Archaea and Eukarya. The nucleotides present at positions 965, 966, 967, 968 and 971, however, are only conserved and unique within each domain. All organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. Biochemical and structure studies have placed this loop near the P site and have shown it to be involved in the decoding process and in binding the antibiotic tetracycline. To identify the functional components of this rRNA hairpin, the eight nucleotides of the 970 loop of helix 31 were subjected to saturation mutagenesis and 107 unique functional mutants were isolated and analyzed. Non-random nucleotide distributions were observed at each mutated position among the functional isolates. Nucleotide identity at positions 966 and 969 significantly affects ribosome function. Ribosomes with single mutations of m 2 G966 or m 5 C967 produce more protein in vivo than wild-type ribosomes. Over-expression of initiation factor 3 (IF3) specifically restored wild-type levels of protein synthesis to the 966 and 967 mutants, suggesting that modification of these residues is important for IF3 binding and for the proper initiation of protein synthesis.

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Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs

Cited by 81 publications

References 47 publications

Modulation of ribosomal recruitment to 5′‐terminal start codons by translation initiation factors IF2 and IF3

Modulation of ribosomal recruitment to 5′‐terminal start codons by translation initiation factors IF2 and IF3

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

Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA

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