Novel Structural and Functional Aspects of Translational Initiation Factor IF2

Spurio, Roberto; Severini, M.; Teana, Anna La; Canonaco, Maria A.; Pawlik, R. T.; Gualerzi, Claudio O.; Pon, Cynthia L.

doi:10.1007/978-1-4615-2407-6_23

Cited by 21 publications

(35 citation statements)

References 35 publications

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“…The B. stearothermophilus IF2 C domain was prepared as previously described [6,7]. Site-directed mutagenesis of Cys-668 and Cys-714 were performed by oligonucleotide-directed mutagenesis using thè altered sites in vitro mutagenesis' protocol (Promega) and the mutations con¢rmed by DNA sequencing.…”

Section: Cloning Expression and Puri¢cation Methodsmentioning

confidence: 99%

“…Additional roles of IF2 in other cellular processes such as transcription [3] and protein secretion [4] have been proposed. To perform its function in translation, IF2 interacts with fMet-tRNA fMet through its C-terminal (24.5 kDa) domain (IF2 C) and with GTP/GDP and 50S ribosomal subunits through its central (40 kDa) G-domain [5,6]. Interaction with 30S is also essential, yet the localization of the active site responsible for this function remains elusive.…”

Section: Introductionmentioning

confidence: 99%

“…Interaction with 30S is also essential, yet the localization of the active site responsible for this function remains elusive. Both G-and C-domains were obtained as recombinant proteins by genetic manipulation of B. stearothermophilus infB and expression in Escherichia coli [6]. Recently, our e¡orts have been concentrated on the attempt to elucidate the structure of the fMet-tRNA fMet binding domain of IF2 (IF2 C) and the molecular nature of its interaction with the initiator tRNA [7,8]; insofar as this interaction is eubacteria-speci¢c and vitally important for cell survival, it could also represent an ideal target for the rational design of a new class of antibiotics which are in high demand.…”

Section: Introductionmentioning

confidence: 99%

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The fMet‐tRNA binding domain of translational initiation factor IF2: role and environment of its two Cys residues

et al. 1999

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Mutations of the cysteines (positions 668 and 714) were generated in the IF2 C domain of Bacillus stearothermophilus translation initiation factor IF2. The corresponding proteins were characterized functionally and structurally. Most (yet not all) amino acid replacements at both positions resulted in severe reduction of the fMet-tRNA binding activity of IF2 C without grossly altering its structure. Our work demonstrates that: (a) both Cys residues are buried within an hydrophobic core and not accessible to protonation or chemical substitution, (b) neither Cys is functionally essential and (c) both Cys residues are located near the active site, probably without participating directly in fMet-tRNA binding.z 1999 Federation of European Biochemical Societies.

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Section: Cloning Expression and Puri¢cation Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The fMet‐tRNA binding domain of translational initiation factor IF2: role and environment of its two Cys residues

et al. 1999

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“…It is relatively stable to protease digestion. Sequence comparison of the C-terminal parts of different prokaryotic IF2 proteins reveals high similarity (Spurio et al, 1993). IF2-C alone (24 kDa) can complex initiator fMettRNA fMet .…”

Section: Introductionmentioning

confidence: 99%

Preliminary characterization by X-ray diffraction and Raman spectroscopy of a crystalline complex ofBacillus stearothermophilusinitiation factor 2 C-domain and fMet-tRNA^fMet

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Krafft

Welfle

et al. 1999

Acta Crystallogr D Biol Cryst

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Bacillus stearothermophilus translation initiation factor 2 (IF2) specifically binds initiator fMet‐tRNAfMet and positions it into the ribosomal peptidyl site in the course of the initiation of protein biosynthesis. The isolated C‐terminal domain of IF2 is capable of binding fMet‐tRNAfMet, as shown by RNase A and hydrolysis protection experiments. In the presence of fMet‐tRNAfMet, the IF2 C‐domain yielded orthorhombic crystals of space group I222 (I212121) diffracting to 3.4 Å resolution. The existence of equimolar amounts of tRNA and protein in the crystals was proven by Raman spectroscopy. The observed unit cell suggests the presence of two IF2 C‐domain–fMet‐tRNAfMet complexes per asymmetric unit of the crystal.

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“…It has been observed that the binding of IF2 to the ribosomal initiation complex in a thermophile organism has a greater stability compared to E. coli (38). Thus, T. thermophilus IF2 may not require a speci c region of its structure with high af nity for the ribosome.…”

Section: Recognition Of the Ribosome By If2mentioning

confidence: 99%

Macromolecular Mimicry in Translation Initiation: A Model for the Initiation Factor IF2 on the Ribosome

et al. 2000

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SummaryProtein biosynthesis in bacteria is controlled by a number of translation factors. Recent data based on comparison of sequence and structure data of translation factors have established a novel hypothesis for their interaction with the ribosome: initiation, elongation, and termination factors may use a common or partly overlapping binding site on the ribosome in a process of macromolecular mimicry of an A-site-bound tRNA. This paper reviews structural knowledge and tRNA macromolecular mimicry involvement of translation initiation factor IF2. Furthermore, a model is proposed for the factor and its interaction with the ribosome during the formation of the translation initiation complex. IUBMB Life, 50: 347 -354, 2000

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Novel Structural and Functional Aspects of Translational Initiation Factor IF2

Cited by 21 publications

References 35 publications

The fMet‐tRNA binding domain of translational initiation factor IF2: role and environment of its two Cys residues

The fMet‐tRNA binding domain of translational initiation factor IF2: role and environment of its two Cys residues

Preliminary characterization by X-ray diffraction and Raman spectroscopy of a crystalline complex ofBacillus stearothermophilusinitiation factor 2 C-domain and fMet-tRNA^fMet

Macromolecular Mimicry in Translation Initiation: A Model for the Initiation Factor IF2 on the Ribosome

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Novel Structural and Functional Aspects of Translational Initiation Factor IF2

Cited by 21 publications

References 35 publications

The fMet‐tRNA binding domain of translational initiation factor IF2: role and environment of its two Cys residues

The fMet‐tRNA binding domain of translational initiation factor IF2: role and environment of its two Cys residues

Preliminary characterization by X-ray diffraction and Raman spectroscopy of a crystalline complex ofBacillus stearothermophilusinitiation factor 2 C-domain and fMet-tRNAfMet

Macromolecular Mimicry in Translation Initiation: A Model for the Initiation Factor IF2 on the Ribosome

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Preliminary characterization by X-ray diffraction and Raman spectroscopy of a crystalline complex ofBacillus stearothermophilusinitiation factor 2 C-domain and fMet-tRNA^fMet