Characterization of the hybridization between purified 16S and 23S ribosomal ribonucleic acid and ribosomal deoxyribonucleic acid from Escherichia coli.

An assay to distinguish specifically between 16S and 23S ribosomal ribonucleic acid (rRNA) has been developed. The assay involves hybridization of radioactive rRNA to deoxyribonucleic acid (DNA) from lambdailv5 transducing phage, which carries an rRNA transcription unit. Radioactive 16S or 23S rRNA can be specifically and completely competed from hybrids by using highly purified nonradioactive 16S or 23S competitor RNA, respectively. The preparation and purification of 16S rRNA and 23S rRNA are described in detail. The hybridization assay is extremely sensitive and efficient; 65 to 70% or more of the input radioactivity hybridizes to specific DNA in the absence of homologous competitor RNA, and at saturation virtually all of the specific DNA sequences are hybridized to rRNA. The results indicate that: (i) the 16S rRNA and 23S rRNA prepared as described are greater than 99% pure, (ii) 16S RRNA and 23S rRNA hybridize with equal efficiency and in equal molar amounts of lambdailv5 DNA; (iii) at saturation, about one molecule of 16S and one molecule of 23S rRNA are hybridized per genome equivalent of lambda ilv 5 DNA; (iv) essentially no cross-hybridization occurs between 16S and 23S rRNA; and (v) the sequence homology between 16S and 23S rRNA is negligible.