Isolation and characterization of certain ribosomal domains : the 5S RNA-protein domain from Escherichia coli and the 'A' protein domain from wheat germ
Date
1982
Authors
Watt, Paul William
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Abstract
Ribosomes are believed to be composed of many structurally and functionally important, protein-protein and RNA-protein domains. Two of these domains have been investigated. These are the 5S RNA-protein domain and the ribosomal 'A' protein domain.
(i) An attempt has been made to isolate the 5S RNA-protein domain from the large ribosomal subunit of the eubacterium Escherichia coli, as the first step in a project to isolate this domain from an archaebacterial source. E. coli 50S subunits were subjected to a low concentration of Mg²⁺ (2 mM), EDTA•Na₂(10 mM) and NH₄Cl (1 M), (A. Liljas, unpublished), as a possible method to remove the 5S RNA protein complex from the ribosome. The suspension was centrifuged at 35,000 rpm for 15 hours in a Beckman Ti60 rotor and the supernatant obtained was passed through a 5-20% sucrose gradient. A fraction was obtained which contained several r-proteins and 5S RNA. In an attempt to purify the complex. further, the fraction was passed through an S200 column. Although evidence suggests that a complex was obtained, attempts to identify the composition were not successful.
(ii) An attempt was also made to isolate the ribosomal 'A' protein domain, (equivalent to EL7/EL12-EL10 from E. coli), from the large subunit of wheat germ ribosomes, and to isolate and characterize its individual components. A putative complex has been found , of molecular weight 58, 000, containing three r-proteins with molecular weights 15, 000, 13, 700 and 32, 000 respectively. The N-terminal portions of each of these r-proteins has been sequenced. One protein (protein 8) was identified by its sequence as the ribosomal 'A' protein , while another (protein 7) has no homology with the N-terminal portion to protein 8. The third protein (protein 10) has been obtained and differs substantially from the other two proteins in molecular weight, but shows an identical N-terminal amino acid sequence to protein 7.