Altered discrimination of start codons and initiator tRNAs by mutant initiation factor 3

O’Connor, Michael B.; Gregory, Steven T.; RajBhandary, Uttam L.; Dahĺberg, Albert E.

doi:10.1017/s1355838201010184

Cited by 49 publications

(56 citation statements)

References 41 publications

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“…Mutations in IF3 generally derepress spurious initiation by fivefold to 40-fold (Table 1; (Sacerdot et al 1996;Sussman et al 1996;O'Connor et al 2001), while the effects of G1338A are twofold or less (Table 1). Furthermore, the ability of IF3 to discriminate the start codon does not depend on the Type II ''G-minor'' interaction, because the degree of repression by IF3 (infC+ versus infC362) is comparable for ribosomes harboring either G or A at position 1338 (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…Nterminal sequencing of polypeptide products of spurious initiation events indicated that, in infC and wild-type strains, translation began with the initiator tRNA (O'Connor et al 1997(O'Connor et al , 2001). Moreover, spurious initiation was observed specifically from codons termed Class IIA (i.e., AUA, AUC, AUU, ACG, and CUG) (Sacerdot et al 1996;Sussman et al 1996), which differ from cognate start codons (AUG, GUG, or UUG) at only one position.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of 16S rRNA mutations that decrease the fidelity of translation initiation

Qin¹,

Abdi²,

Fredrick³

2007

RNA

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In bacteria, initiation of translation is kinetically controlled by factors IF1, IF2, and IF3, which work in conjunction with the 30S subunit to ensure accurate selection of the initiator tRNA (fMet-tRNA fMet ) and the start codon. Here, we show that mutations G1338A and A790G of 16S rRNA decrease initiation fidelity in vivo and do so in distinct ways. Mutation G1338A increases the affinity of tRNA fMet for the 30S subunit, suggesting that G1338 normally forms a suboptimal Type II interaction with fMettRNA fMet . By stabilizing fMet-tRNA fMet in the preinitiation complex, G1338A may partially compensate for mismatches in the codon-anti-codon helix and thereby increase spurious initiation. Unlike G1338A, A790G decreases the affinity of IF3 for the 30S subunit. This may indirectly stabilize fMet-tRNA fMet in the preinitiation complex and/or promote premature docking of the 50S subunit, resulting in increased levels of spurious initiation.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Characterization of 16S rRNA mutations that decrease the fidelity of translation initiation

Qin¹,

Abdi²,

Fredrick³

2007

RNA

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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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“…The other involves an error in start codon selection, where fMet-tRNA pairs with a codon other than the correct start codon. N-terminal sequencing of products of spurious initiation events suggests that translation in these cases begins with fMet-tRNA (O'Connor et al 1997(O'Connor et al , 2001). Furthermore, spurious initiation has been observed to occur from codons termed Class IIA (e.g., AUA, AUC, AUU, ACG, and CUG), which differ from the cognate AUG at only one position (Sacerdot et al 1996;Sussman et al 1996).…”

Section: Introductionmentioning

confidence: 99%

Role of helix 44 of 16S rRNA in the fidelity of translation initiation

Qin

Liu²,

Devaraj³

et al. 2012

RNA

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The molecular mechanisms that govern translation initiation to ensure accuracy remain unclear. Here, we provide evidence that the subunit-joining step of initiation is controlled in part by a conformational change in the 1408 region of helix h44. First, chemical probing of 30S initiation complexes formed with either a cognate (AUG) or near-cognate (AUC) start codon shows that an IF1-dependent enhancement at A1408 is reduced in the presence of AUG. This change in reactivity is due to a conformational change rather than loss of IF1, because other portions of the IF1 footprint are unchanged and high concentrations of IF1 fail to diminish the reactivity difference seen at A1408. Second, mutations in h44 such as A1413C stimulate 50S docking and cause reduced reactivity at A1408. Third, streptomycin, which has been shown by Rodnina and coworkers to stimulate 50S docking by reversing the inhibitory effects of IF1, also causes reduced reactivity at A1408. Collectively, these data support a model in which IF1 alters the A1408 region of h44 in a way that makes 50S docking unfavorable, and canonical codon-anticodon pairing in the P site restores h44 to a docking-favorable conformation. We also find that, in the absence of factors, the cognate 30S d AUG d fMet-tRNA ternary complex is >1000-fold more stable than the nearcognate 30SdAUCdfMet-tRNA complex. Hence, the selectivity of ternary complex formation is inherently high, exceeding that of initiation in vivo by more than 10-fold.

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Altered discrimination of start codons and initiator tRNAs by mutant initiation factor 3

Cited by 49 publications

References 41 publications

Characterization of 16S rRNA mutations that decrease the fidelity of translation initiation

Characterization of 16S rRNA mutations that decrease the fidelity of translation initiation

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

Role of helix 44 of 16S rRNA in the fidelity of translation initiation

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