Ribosomal Interaction of Bacillus stearothermophilus Translation Initiation Factor IF2: Characterization of the Active Sites
| Autor: | Carlotta Ferrara, Pohl Milón, Jerneja Tomšic, Enrico Caserta, Alessandra Rocchetti, Claudio O. Gualerzi, Anna La Teana, Cynthia L. Pon, Attilio Fabbretti |
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| Rok vydání: | 2010 |
| Předmět: |
Ribosomal Interaction
Mutant GTPase Prokaryotic Initiation Factor-2 Biology medicine.disease_cause Ribosome Protein Structure Secondary Geobacillus stearothermophilus Structural Biology Catalytic Domain Escherichia coli Ribosome Subunits medicine Initiation factor Molecular Biology Sequence Deletion Genetics Mutation Genetic Complementation Test Dipeptides Ribosomal RNA Null allele Kinetics Amino Acid Substitution Genes Bacterial Protein Biosynthesis Mutant Proteins Ribosomes Protein Binding |
| Zdroj: | Journal of Molecular Biology. 396:118-129 |
| ISSN: | 0022-2836 |
| Popis: | InfB-encoded translation initiation factor IF2 contains a non-conserved N-terminal domain and two conserved domains (G and C) constituted by three (G1, G2 and G3) and two (C1 and C2) sub-domains. Here, we show that: (i) Bacillus stearothermophilus IF2 complements in vivo an Escherichia coli infB null mutation and (ii) the N-domain of B. stearothermophilus IF2, like that of E. coli IF2, provides a strong yet dispensable interaction with 30 S and 50 S subunits in spite of the lack of any size, sequence or structural homology between the N-domains of the two factors. Furthermore, the nature of the B. stearothermophilus IF2 sites involved in establishing the functional interactions with the ribosome was investigated by generating deletion, random and site-directed mutations within sub-domains G2 or G3 of a molecule carrying an H301Y substitution in switch II of the G2 module, which impairs the ribosome-dependent GTPase activity of IF2. By selecting suppressors of the dominant-lethal phenotype caused by the H301Y substitution, three independent mutants impaired in ribosome binding were identified; namely, S387P (in G2) and G420E and E424K (in G3). The functional properties of these mutants and those of the deletion mutants are compatible with the premise that IF2 interacts with 30 S and 50 S subunits via G3 and G2 modules, respectively. However, beyond this generalization, because the mutation in G2 resulted in a functional alteration of G3 and vice versa, our results indicate the existence of extensive "cross-talking" between these two modules, highlighting a harmonic conformational cooperation between G2 and G3 required for a functional interaction between IF2 and the two ribosomal subunits. It is noteworthy that the E424K mutant, which completely lacks GTPase activity, displays IF2 wild-type capacity in supporting initiation of dipeptide formation. |
| Databáze: | OpenAIRE |
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