The action of snake venom phosphodiesterase on liver ribosomal ribonucleic acids

Abstract

The stepwise hydrolysis of rat-liver ribosomal RNA's (rRNA) with snake venom phosphodiesterase (EC 3.1.4.1) has been studied. The following results have been obtained: 1. 1. Hydrolysis of poly (U), poly (A) and equimolar mixtures of them proceeds to completion and yields pU and pA as the only major products of the reaction. Secondary structure in poly (U) · poly (A) has no detectable influence on the rate and the extent of hydrolysis. The substrate (Ap) nCp is resistant to the action of this enzyme, while (Ap) nC is rapidly degraded to give pA and pC. 2. 2. As shown by agar-gel electrophoresis, the liver rRNA studied is constituted of homogeneous 28-S and 18-S RNA molecules. They display secondary structure transitions typical for native rRNA's. The preparations studied are virtually free from endonucleolytic contaminants. 3. 3. Hydrolysis of rRNA's with venom phosphodiesterase goes to completion and yields pG, pC, pA and pU as the only major products. Analysis by determination of the nucleosides originating from chain terminals during hydrolysis, shows that the endonuclease contaminants in this system may account for no more than 1 to 2 internal breaks per RNA molecule. 4. 4. The molar ratio of the mononucleotides liberated on stepwise hydrolysis of liver rRNA's with venom phosphodiesterase varies with progress of enzymatic degradation. The product obtained at early stages of enzyme action has a higher content of pA and pU and a lower content of pG and pC. The same correlation is observed with 18-S rRNA and partly with 28-S rRNA. It is suggested that in rRNA the segment near the 3′-end of the chain contains more pA and pU and less pG and pC as compared with the segment near the 5′-end of the chain. 5. 5. Labeling in vivo of free uridine 5′-phosphates and cytidine 5′-phosphates with [6- 14C]orotic acid shows with both nucleotides a maximum incorporation at 40 min. Ribosomal RNA's labeled for 90 min in vivo with [6- 14C]orotic acid were analysed by partial degradation with venom phosphodiesterase and determination of the radioactivity of uridylic acid residues. The labeling of 18-S RNA is higher than of 28-S RNA. Enzymatic liberation of labeled uridylic acid from 18-S and 28-S RNA is non-uniform. With both 18-S and 28-S RNA, uridylic acid liberated at the initial stages of enzyme action is more highly labeled than uridylic acid from the remaining RNA fragments.