Abstract
In vitro reconstitution systems of ribosomal subunits from free ribosomal RNA and ribosomal proteins are helpful tool for studies on the structure, function and assembly of ribosome. Using this system mutant or modified ribosomal proteins or ribosomal RNA can be incorporated into ribosomal subunits for studying ribosome structure and function. Developing the protocol for reconstitution of 30S subunits from an extreme thermophilic bacterium Thermus thermophilus can be beneficial especially for structural studies, as proteins and nucleic acids from this organism are very stable and crystallize easier than those from mesophilic organisms.
Key words
- Ribosome
- 30S subunit
- In vitro reconstitution
- Thermus thermophilus
- Ribosomal proteins
- RNA
- Polyamines
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References
Traub P, Nomura M (1968) Structure and function of E. coli ribosomes. V. Reconstitution of functionally active 30S ribosomal particles from RNA and proteins. Proc Natl Acad Sci U S A 59:777–784
Mizushima S, Nomura M (1970) Assembly mapping of 30S ribosomal proteins from E. coli. Nature 226:1214
Held WA, Mizushima S, Nomura M (1973) Reconstitution of Escherichia coli 30S ribosomal subunits from purified molecular components. J Biol Chem 248:5720–5730
Culver GM, Noller HF (1999) Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins. RNA 5:832–843
Culver GM, Noller HF (2000) In vitro reconstitution of 30S ribosomal subunits using complete set of recombinant proteins. Methods Enzymol 318:446–460
Krzyzosiak W, Denman R, Nurse K, Hellmann W, Boublik M, Gehrke CW, Agris PF, Ofengand J (1987) In vitro synthesis of 16S ribosomal RNA containing single base changes and assembly into a functional 30S ribosome. Biochemistry 26:2353–2364
Cohlberg JA, Nomura M (1976) Reconstitution of Bacillus stearothermophilus 50S ribosomal subunits from purified molecular components. J Biol Chem 251:209–221
Fahnestock SR (1979) Reconstitution of active 50S ribosomal subunits from Bacillus licheniformis and Bacillus subtilis. Methods Enzymol 59:437–443
Sanchez ME, Urena D, Amils R, Londei P (1990) In vitro reassembly of active large ribosomal subunits of the halophilic archaebacterium Haloferax mediterranei. Biochemistry 29:9256–9261
Londei P, Teixido J, Acca M, Cammarano P, Amils R (1986) Total reconstitution of active large ribosomal subunits of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Nucleic Acids Res 14:2269–2285
Oshima T (1974) Comparative studies on biochemical properties of an extreme thermophile, Thermus thermophilus HB 8 (author’s transl). Seikagaku 46:887–907
Trakhanov SD, Yusupov MM, Agalarov SC, Garber M, Ryazancev SN, Tischenko SV, Shirokov VA (1987) Crystallization of 70S ribosomes and 30S ribosomal subunits from Thermus thermophilus. FEBS Lett 220:319–322
Garber M, Agalarov C, Eliseikina I, Tischenko S, Shirokov V, Yusupov M, Reshetnikova L, Trakhanov S, Tukalo M, Yaremchuk A (1991) Purification and crystallization of components of the protein-synthesizing system from Thermus thermophilus. J Cryst Growth 110:228–236
Yusupova G, Yusupov M, Spirin A, Ebel JP, Moras D, Ehresmann C, Ehresmann B (1991) Formation and crystallization of Thermus thermophilus 70S ribosome/tRNA complexes. FEBS Lett 290:69–72
Igarashi K, Kashiwagi K, Kishida K, Watanabe Y, Kogo A, Hirose S (1979) Defect in the split proteins of 30-S ribosomal subunits and under-methylation of 16-S ribosomal RNA in a polyamine-requiring mutant of Escherichia coli grown in the absence of polyamines. Eur J Biochem 93:345–353
Kakegawa T, Hirose S, Kashiwagi K, Igarashi K (1986) Effect of polyamines on in vitro reconstitution of ribosomal subunits. Eur J Biochem 158:265–269
Igarashi K, Kashiwagi K, Kishida K, Kakegawa T, Hirose S (1981) Decrease in the S1 protein of 30-S ribosomal subunits in polyamine-requiring mutants of Escherichia coli grown in the absence of polyamines. Eur J Biochem 114:127–131
Khaitovich P, Tenson T, Kloss P, Mankin AS (1999) Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA. Biochemistry 38:1780–1788
Oshima T (1975) Thermine: a new polyamine from an extreme thermophile. Biochem Biophys Res Commun 63:1093–1098
Gogia ZV, Yusupov MM, Spirina TN (1986) Structure of Thermus thermophilus ribosomes method of isolation and purification of the ribosomes. Molekul Biol (USSR) 20:519–526
Acknowledgements
We are grateful to A.S. Spirin for his support of early stages on the study of Thermus thermophilus ribosomes; V.A. Shirokov for stimulating discussions. This work was supported by Russian Academy of Science.
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Agalarov, S., Yusupov, M., Yusupova, G. (2016). Reconstitution of Functionally Active Thermus thermophilus 30S Ribosomal Subunit from Ribosomal 16S RNA and Ribosomal Proteins. In: Ennifar, E. (eds) Nucleic Acid Crystallography. Methods in Molecular Biology, vol 1320. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2763-0_19
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DOI: https://doi.org/10.1007/978-1-4939-2763-0_19
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