Abstract
Isolation of ribosomal particles is an essential step in the study of ribosomal components as well as in the analysis of trans-acting factors that interact with the ribosome to regulate protein synthesis and modulate the expression profile of the cell in response to different environmental conditions. In this protocol, we describe a procedure for the isolation of 70S ribosomes from the unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We have successfully used this protocol in our study of the cyanobacterial ribosomal-associated protein LrtA, which is a homologue of bacterial HPF (hibernation promoting factor) (Galmozzi et al., 2016).
Keywords: Ribosome, Sucrose gradients, Synechocystis, Cyanobacteria, Photosynthetic prokaryotes
Background
Few biochemical studies have been reported for cyanobacterial ribosomes. Anabaena variabilis strain M3 (PCC 7118, ATCC 27892) 70S ribosomal particles have been isolated by differential centrifugation and then, ribosomal proteins were analysed by two-dimensional electrophoresis (Sato et al., 1998). Ribosomes have also been prepared from Synechococcus sp. PCC 6301 cells using a protocol combining differential centrifugation and sucrose step gradients (Sugita et al., 2000). Fractionation of cell extracts by differential centrifugation has also been employed in the preparation of ribosomal samples for the development of an in vitro translation system in different Synechococcus strains (Mutsuda and Sugiura, 2006). The method described here for Synechocystis, based on the one described for Synechococcus (Sugita et al., 2000), allows purification of ribosomal particles using ultracentrifugation of linear sucrose gradients.
Materials and Reagents
Equipment
Procedure
An overview of the whole protocol, divided in four steps, is shown in Figure 1. Figure 1. Scheme of the complete procedure described in the protocol
Notes
Nowadays the analog signal from the ISCO UA-6 detector can be transformed into a digital signal using a Bus-Powered Multifunction DAQ USB Device (NI USB-6008) from National Instruments.
Recipes
Note: All solutions are stored at 4 °C.
Acknowledgments
This protocol was adapted and modified from previously published studies by Sugita et al. (2000). We are grateful to Dr. J. de la Cruz for his expert advice, helpful discussions and a critical reading of the manuscript. We thank M. Roldán for technical assistance. This work was supported by Junta de Andalucía (grant P07-CVI-02792 and group BIO-284) and Spanish Ministerio de Economía y Competitividad (MINECO) and Fondo Social Europeo (FSE) (grant BFU2013-41712-P).
References
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