The Use of Nucleoside Phosphotransferase and (³²P) p-Nitrophenyl Phosphate in the Determination of the 5′-Linked Termini of Ribosomal RNA

Abstract

(2,3,16). Since the artificial substrate p-nitrophenyl phosphate could serve as a donor, and it appeared that radioactive p-nitrophenyl phosphate could be synthesized, it was felt that the enzymatic synthesis of radioactive nucleotides utilizing (32P) p-nitrophenyl phosphate and the enzyme could prove to be tuseful in the identification of nucleosides.In studies on the terminal groups of ribosomal RNA it is either necessarv to have large quantities of RNA available or to utilize techniques capable of detecting small amounts of the terminal groups. Often it is not feasible to obtain large quantities of material and con,sequently the latter method must be pursued. This paper then reports the isolation and partial purification of nucleoside phosphotransferase from carrot leaves, the synthesis of (32p) For a discussion concerning the formation, structure, and function of ribosomal RNA the reader is referred to the reviews of Madison (12) and Osawa (16). The various aspects of determining the nucleic acid sequences and termini also have been recently reviewed by RajBhandary and Stuart (17). Several papers have been published dealing with the identification of the 5'-linked termini of ribosomal RNA (7,9,10,11,13,15). From the studies on these termini, the results most consistently obtained are that there is a preferential termination of ribosomal RNA by adenosine and uridine. Exceptions to the rule are the investigations on wheat germ ribosomal RNA (9, 11) from whence equal amounts of each of the 4 nucleosides were isolated. Recently, evidence was presented from this laboratory which indicated that adenosine and uridine are the predominant 5'-linked termini of wheat germ ribosomal RNA as well as ribosomal RNA from 4 other higher plants and a fungus (6). Materials and Methods

The 5'-linked termini of plant ribosomal ribonucleic acids

“…ferase isolated from carrot leaves, as described by Becker and Pollard (1969). Nucleosides were incubated for 6 hr, at 37°, with 0.1 ml of 0.1 m sodium acetate (pH 5.1), 50 µ of [32P]p-nitrophenyl phosphate (15.8 mg/ml, activity = 10,000 cpm^g), and 0.1 ml of a nucleoside phosphotransferase preparation.…”