163b] Amino acid incorporation mediated by ribosomal RNA and soluble RNA

Abstract

Publisher Summary This chapter describes the process of amino acid incorporation mediated by ribosomal RNA and soluble RNA. Ribosomes are obtained free of messenger by twice centrifuging at 40,000 rpm for 3 hours in Tris 0.01 M , pH 7.8, magnesium acetate, l0 –4 M . The 50 S and 30 S subunits are obtained, and the messenger is dissociated from the ribosomes and degraded by the traces of nuclease present. Purified 16 S and 23 S RNA are obtained by sucrose gradient separation of the 50 S and 30 S ribosomes or the mixture obtained by phenol extraction of the ribosome subunits. To conveniently purify soluble RNA, ribosomes are removed from an S 30 fraction by centrifuging at 40,000 rpm for 2 hours in Tris, 0.01 M (pH 7.8), and magnesium acetate, 0.01 M . Messenger remains attached to ribosomes and is largely removed, and phenol extraction of the supernatant yields crude soluble RNA. The addition of streptomycin on similar antibiotics is not sufficient to obtain optimal template activity. Both ribosomal and soluble RNA must be heat treated to obtain approximately one break in the molecule. This may be achieved by boiling in 0.01 M phosphate buffer (pH 6.8) for about 4 minutes for ribosomal RNA and about 1 hour for soluble RNA.