Binding of Escherichia coli protein synthesis initiation factor IF1 to 30S ribosomal subunits measured by fluorescence polarization

Zucker, Frank; Hershey, John W.B.

doi:10.1021/bi00360a031

Cited by 42 publications

(53 citation statements)

References 22 publications

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“…Their coordinated function in several defined steps leads to the formation of a 70s ribosome that carries the mRNA and the forniylmethionine-(Met)-tRNA in the correct positions, thereby enabling entry into the elongation phase of translation (for a review, see Gualerzi and Pon, 1990). IF1 is involved in the association/dissociation processes of the ribosomal subunits (Godefroy-Colburn et al, 1975) and stimulates IF2 binding to the 30s subunit (Zucker and Hershey, 1986). Recently, it was proposed that IF1 mimics the A-site-bound tRNA, thereby preventing premature binding of an aminoacyl-tRNA to the 30s A site (Moazed et al, 1995).…”

Section: Identification and Purification Of Translation Initiation Famentioning

confidence: 99%

Identification and Purification of Translation Initiation Factor 2 (IF2) from Thermus thermophilus

Vornlocher

Scheible

Faulhammer

et al. 1997

European Journal of Biochemistry

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Translation initiation factor 2 (IF2) is one of three protein factors required for initiation of protein synthesis in eubacteria. The protein is responsible for binding the initiator tRNA to the ribosomal P site. IF2 is a member of the GTP/GDP-binding protein superfamily. In the extreme thermophilic bacterium Themus rhermophilus, IF2 was identified as a 66-kDa protein by affinity labeling and immunoblotting. The protein was purified to homogeneity. The specific activity indicates a stoichiometric IF2-mediated binding of formylmethionine-tRNA to 70s ribosomes. The N-terminal amino acid sequences of the intact protein and of two proteolytic fragments of 25 kDa and 40 kDa were determined. Comparison with other bacterial IF2 sequences indicates a similar domain architecture in all bacterial IF2 proteins.

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Section: Identification and Purification Of Translation Initiation Famentioning

confidence: 99%

Identification and Purification of Translation Initiation Factor 2 (IF2) from Thermus thermophilus

Vornlocher

Scheible

Faulhammer

et al. 1997

European Journal of Biochemistry

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“…IF1 binds to the 30S subunit in a 1:1 ratio and this binding is strengthened in the presence of IF2 and IF3 [5,6]. On the other hand, the activities of IF2 and IF3 in the formation of the pre-initiation complex are stimulated when IF1 is present [7,8].…”

Section: Introductionmentioning

confidence: 99%

In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level

2005

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Edited by Lev KisselevAbstract The influence in vivo of mutated forms of translation initiation factor (IF1) on the expression of the lacZ or 3A 0 reporter genes, with different initiation and/or +2 codons, has been investigated. Reporter gene expression in these infA(IF1) mutants is similar to the wild-type strain. The results do not support the longstanding hypothesis that IF1 could perform discriminatory functions while blocking the aminoacyl-tRNA acceptor site (A-site) of the ribosome. One cold-sensitive IF1 mutant shows a general overexpression, in particular at low temperatures, of both reporter genes at the protein but not mRNA level.

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“…IF1 enhances the rates of 70S ribosome dissociation and subunit association but does not change the equilibrium position (10). It alone binds poorly to 30S ribosomal subunits, but when added with IF2 both factor-binding affinities are increased (32). IF1 stimulates IF2-dependent fMet-tRNAf binding to 30S or 70S ribosomes in the presence of mRNA.…”

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confidence: 96%

Translation initiation factor IF1 is essential for cell viability in Escherichia coli

Cummings

Hershey

1994

J Bacteriol

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Translation initiation factor IF1 is a highbly conserved element of the prokaryotic translational apparatus. It has been demonstrated earlier that the factor stimulates in vitro the initiation phase of protein synthesis. However, no mutation in its gene, infA, has been identified, and a role for IF1 in translation has not been demonstrated in vivo. To elucidate the function of IFi and determine if the protein is essential for cell growth, the chromosomal copy of infA was disrupted. Cell viability is maintained only when infA is expressed in trans from a plasmid, thereby demonstrating that WI1 is essential for cell growth in Escherichia coli. Cells depleted of IF1 exhibit few polsomes, suggesting that IFI functions in the initiation phase of protein synthesis.Initiation of protein synthesis in prokaryotes involves formation of a 30S preinitiation complex in which fMet-tRNA interacts with the initiation codon of an mRNA bound to the surface of the 30S ribosomal subunit. The efficiency and fidelity of formation of the 30S preinitiation complex is promoted by three initiation factors; IF1, IF2, and IF3. The properties and mechanism of action of the initiation factors have been reviewed recently (13). All three initiation factors bind to the 30S ribosomal subunit in the absence of other translational components. IF2 binds GTP and fMet-tRNAf, thereby ensuring that the tRNA bound to the 30S subunit is the initiator tRNA, fMet-tRNAf. 1F3 plays at least two roles: it acts as an antiassociation factor to maintain the ribosomal subunits in a dissociated state, and it promotes the selection of the initiator fMet-tRNA by monitoring the tRNA anticodon stem-loop region and the correctness of the anticodon-initiator codon interaction on the ribosome. The function of IF1 is less clear, since no specific role has yet been assigned to this protein.IF1 is the smallest of the initiation factors (Mr,8,100) and consists of 71 amino acid residues. The gene for IF1, infA, has been cloned, sequenced, and mapped to about 20 min on the Escherichia coli chromosome (25). The infA gene is transcribed by two promoters to yield two sizes of monocistronic mRNAs both ending at the same terminator (8). Recently, homologous genes have been identified in Bacillus subtilis and in the chloroplasts of several plants (4,23,28). The high degree of homology indicates that IF1 is a conserved protein and suggests that it plays an important role in the cell. However, our knowledge of the function of IF1 comes exclusively from a number of in vitro assays for translation. IF1 enhances the rates of 70S ribosome dissociation and subunit association but does not change the equilibrium position (10). It alone binds poorly to 30S ribosomal subunits, but when added with IF2 both factor-binding affinities are increased (32). IF1 stimulates IF2-dependent fMet-tRNAf binding to 30S or 70S ribosomes in the presence of mRNA. However, the best demonstration that IF1 truly functions in protein synthesis comes from the observation that ,-galactosidase synthesis in a DNA-l...

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Binding of Escherichia coli protein synthesis initiation factor IF1 to 30S ribosomal subunits measured by fluorescence polarization

Cited by 42 publications

References 22 publications

Identification and Purification of Translation Initiation Factor 2 (IF2) from Thermus thermophilus

Identification and Purification of Translation Initiation Factor 2 (IF2) from Thermus thermophilus

In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level

Translation initiation factor IF1 is essential for cell viability in Escherichia coli

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