The Temperature‐Dependent Enzymatic Breakdown of rRNA of Reticulocytes

Abstract

The influence of temperature on the enzymatic breakdown of rRNA and ribosomal particles from rabbit reticulocytes by ribosomal ribonuclease and phosphatase was studied. Nucleosides were formed after the initial attack of ribonuclease on single-stranded regions of the RNA.The following conclusions were reached for free RNA: U and C were distributed in a random manner in the single-stranded loops a t 20 "C. Short (A-U)-rich sequences were liberated at 37 "C, the relative share of which appeared to be higher in the 28-5 RNA. It contained correspondingly longer G-C sequences. Clusters of A-U sequences of greater length than a t 37 "C were melted a t 50 "C. It was proved that only -U-U-U-sequences were split, by analysis of of the 3' and 5' terminal nucleotides of the primary split products of ribonuclease.The behaviour of the particle-bound RNA was modi6ed considerably by interactions with proteins. Some of the cytosines appear to be protected against enzymatic attack a t 20 "C. The double-stranded sections apparently exhibit variation both with regard to the number of bases in the sequence, which ranges from 4 to 10, and their chemical nature. There is extensive clustering of identical bases. The main method for the study of the ribosome has been the analysis of the temperature profiles and titrations, which lends itself only to statistical conclusions.Enzynzes. Ribonuclease (EC 2.7.7.16); Phosphatase (EC 3.1.3.1) ; Phosphodiesterase (spleen) (EC 3.1.4.1).Previously we reported temperature-dependent changes in the specifity of enzymatic degradation of free rRNA [12]. The U-rich double-stranded sections were melted between 30 "C and 50 "C and were subject to preferential enzymatic breakdown. The results agree with the model of Cox [ill. The purpose of the present study was to gain additional information from a systematic study of the temperaturedependent enzymatic breakdown of both free rRNA and ribosomal particles. MATERIALS AND METHODSThe ribosomes from reticulocytes were prepared according to a published procedure . To determine the nucleotide composition the tRNA was hydrolyzed for I8 h in 0.3 M KOH a t 25 "C. After neutralization with HC10, and removal of the KClO, by centrifugation the nucleotides were fractionated on a Dowex column (1x8, 100 to 200 mesh, formate form) by elution with formic acid/ sodium formate. They were assayed spectrophotometrically.

Pyrimidine-Rich Oligonucleotides from Rat-Liver Ribosome Surface

“…This fact agrees with the finding of Prehn et al [18], namely, that uridine and cytosinerich sequences were the first to be hydrolysed in rRNA. Thus, the removal of oligonucleotides from the ribosome is similar to the first steps in the digestion of free ribosomal RNA.…”