Characterization of Bacillus stearothermophilus infA and of its product IF1
Introduction
IF1, one of the three factors essential to initiate protein synthesis in bacteria is a β-barrel RNA binding protein (Gualerzi, 2001, Boelens and Gualerzi, 2002, Laursen et al., 2005) with RNA chaperone activity (Croitoru et al., 2006) which binds to the 30S ribosomal subunit (Celano et al., 1988, Carter, 2001) induces a conformational change of the subunit mainly involving helix 44 of 16S rRNA (Carter et al., 2001) and stimulates, together with IF2 and IF3, the formation of both 30S and 70S initiation complexes (30SIC and 70SIC) (Wintermeyer and Gualerzi, 1983, Pon and Gualerzi, 1984). Furthermore, IF1 cooperates with IF3 to maintain a sufficient pool of free subunits (Godefroy-Colburn et al., 1975, Giangrossi et al., 2007) and to ensure initiation fidelity by discriminating against non-canonical 30S initiation complexes (Milon et al., 2008).
Although IF1 is one of the universally conserved translational factors (Kyrpides and Woese, 1998), several attempts to isolate and characterize Bacillus stearothermophilus (Bst) infA and its product BstIF1 had failed in the past in our laboratory, unlike the case of infB (Brombach et al., 1986) and infC (Pon et al., 1989) of this organism which were successfully isolated, characterized and expressed, yielding large amounts of thermophilic IF2 and IF3.
The present isolation, characterization and expression of B. stearothermophilus infA fill this gap, while the consequent availability of purified BstIF1 allows us to use translational components derived from this organism to set up one of the most active initiation tests available for Gram positive bacteria.
Section snippets
Buffers
Buffer I: 10 mM Tris–HCl pH 7.7, 60 mM NH4Cl, 10 mM MgAcetate
Buffer II: 20 mM Bis-Tris pH 6.1, 1 mM EDTA pH 7.0, 10% Glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM benzamidine
Buffer III: 20 mM Tris–HCl pH 7.1, 1 mM EDTA pH 7.0, 10% Glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM benzamidine
TBS Buffer: 0.05 M Tris–HCl (pH 7.6), 0.9% NaCl.
Formation of 30SIC and 70SIC, kinetic experiments by FRET stopped-flow experiments and in vitro translation of 027mRNA were performed as
Isolation, sequencing and cloning of Bst infA
A preliminary genomic comparison of B. subtilis and B. (Geobacillus) stearothermophilus, partially available on line (http://www.genome.ou.edu/bstearo.html) revealed a high degree of sequence and gene organization conservation within the regions chosen for comparison (i.e. the S10, the spc and the alfa operons) (Fig. 1A). However, the available B. stearothermophilus sequence comprised only a limited portion of what seemed to be infA (Fig. 1B). To isolate and characterize infA it was therefore
Acknowledgments
Grants of the Italian MIUR (PRIN 2005) to COG and CLP are gratefully acknowledged.
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