Abstract
The eubacterial ribosome (70S) is a macromolecular complex that is composed of a small (30S) subunit and a large (50S) subunit. The small subunit comprises the 16S ribosomal RNA (rRNA) and more than 20 ribosomal proteins (r-proteins), whereas the large subunit comprises the 23S and 5S rRNAs and more than 30 r-proteins. Bacillus subtilis (B. subtilis) has 57 r-ribosomal protein genes and three rRNAs (16S, 23S and 5S rRNAs). Among them, we identified 21 r-proteins of the small subunit and 31 r-proteins of the large subunit in B. subtilis (Nanamiya et al., 2004). The functions and roles of individual components of the ribosome have not yet been completely clarified. Herein we describe in detail an ultracentrifugation-based protocol for the preparation of 70S ribosomes from exponentially growing cells of B. subtilis.
Keywords: Bacillus subtilis, Ribosome, 70S ribosomes
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Profile of ribosome from crude cell extract by 10-40% sucrose gradient sedimentation. The crude cell extract prepared from the log phase cells (OD600 = 0.2) of wild type strain (168 trpC2) was applied on the top of the 10 to 40% sucrose gradient and centrifuged at 55,000 x g for 17.5 h at 4 °C (e.g. in a HITACHI P40ST rotor at 21,000 rpm, using 10 A260 units per centrifuge tube). The A260 measurement shows as the same profile pattern of A420 measurement, and the peak of 70S ribosome can be determined. When the S100 fraction was subjected to the 10 to 40% sucrose gradient and centrifugation, the peak smaller than 30S was not observed.
Recipes
Acknowledgments
This protocol was adapted from Nanamiya et al. (2004) and Suzuki et al. (2014). This work was supported in part by Grants-in-Aid for Scientific Research (C) (G. A.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
References
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