Isolation of a high specific activity 35S ribosomal RNA precursor from Tetrahymena pyriformis and identification of its 5' terminus, pppAp

Abstract

RNA isolated from starved and refed T. pyriformis pulse labeled with H332PO4 contains an RNA molecule of 2.4 .times. 106 MW which can be identified by polyacrylamide gel electrophoresis. This RNA was shown to be a precursor of the mature 25S rRNA and 17S rRNA by a kinetic analysis of its synthesis, by hybridization competition with pure 25S rRNA and 17S rRNA, and by 2-dimensional fingerprint analysis. The 5''-terminal nucleotide was isolated from the total digestion products of the 35S rRNA precursor and shown to be pppAp by analysis of the products of snake venom phosphodiesterase digestion. Low but significant yields of the 5'' terminus pppAp were isolated from the 17S rRNA, demonstrating that the primary transcript of the rRNA gene in T. pyriformis contains the 17S rRNA at its 5'' end.