The chain termini of polynucleotides formed by limited enzymic fragmentation of wheat embryo ribosomal RNA. Part 1. Studies of snake venom phosphodiesterase

Abstract

The chain termini of polynucleotides formed by limited enzymic fragmentation of wheat embryo ribosomal RNA. Part 1. Studies of snake venom phosphodiesterase. Can. J. Biochem. 46, 81 (1968).Snake venom phosphodiesterase induces about fifteen exonucleolytic cleavages for each endonucleolytic cleavage during the first hour of hydrolysis sf wheat embryo ribosomal RNA, under the conditions of hydrolysis used in this present investigation. The polynucleotide chains in the sibosornaI RNA preparation have am average degree of polymerkation in the neighborhood of 1308 nucleotide residues, and there is a mean of between 5 md 18 endonucleolytic breaks per chain during this airst hour of phosphodiesterase-induced hydrolysis. The cleavages occur widely throughout most of the polynucleotide chains in the ribosomal RNA preparation, as judged by the sharp decrease in mean sedimentation rate which accompanies a limited degree (about 10%) of exonucleolpis of the RNA. Studies of phosphodiestaase-indumd endonucleolysis of wheat embryo soluble RNA are reported, but because of the much lower initial degree of polymerization (about 88 nucleotide residues per polynucleotide chain), the results of endonucleolysis are less pronounced in terms of the proportional increment in chain termini and the proportional decrease of mean sedimentation sate. The endonucleolysis of RNA is discussed in tams of the minor nucleotide components in both ribosomal and soluble RNA, and particular reference is made to pseudouridylate which has been found in relatively high proportion among the chain termini after limited hydrolysis with venom phosphodiesterase.Purified venom phosphodiesterase preparations, devoid of ribonuclease or 5'-naacleotidase contamination, were found to convert nucleoside 2'(3'),5'-diphosphsates to 5'-nucleotides under conditions which had virtually no effect on nucleoside 3'-phosphates or nucleoside %'-phosphates. The possibility that this reaction may be catalyzed by the venom phosphodiesterase itself is discussed.In 1951, Marko and Butler (1) introduced the sodium dodecyl suKate procedure for the isolation of calf thymus DNA', and in the same year, Hurst and Butler (2), succeeded in obtaining snake venom phosphodiesterase preparations, which in concert with pancreas DNase effected an almost quantitative conversion of the highly polymerized DNA to its constituent 5'-deoxyr-ibonucleotides (4). By 1955, Helleiner (4) had established that the hydrolysis catalyzed by venom phosphodiesterase was exonucieoiyiic. Although this exonucleolytic action of venom phosphodiesterase has been exploited to advantage,there have been few studiesdesiped to assess the extent to which venom phosphodiesterase 'Abbreviations : DNA, deoxyribonucleate(s) ; rRNA, ribosomal ribonuclea%e(s) ; sRNA, solublle ribsnucleate(s) ; NxpNp, alkali-stable dinucleotides containing 2'-Qmthylribose in which N is any of the four major riboaaucleosides in RNA and Nx is the 2'-0-methylated derivative of m y sf the four principal nuclmsides; pA, adenosine 5'-phosphate; A2p, adeno...

Pseudouridine in RNA: What, Where, How, and Why

“…More than 30 years ago, McLennan and Lane (76) inferred that W residues are probably concentrated at the junctions between single-stranded and helical regions in tRNA and rRNA. Detailed physicochemical studies (5,6) have now elucidated the consequences of such localization, since con rmed, in stabilizing higher-order structures.…”

Negative charge at the 3'-terminus of oligonucleotides and resistance to venom exonuclease

“…However, there is little evidence in Figure 9 of further dephosphorylation to nucleoside. Several authors (Sulkowski et al, 1963;McLennan and Lane, 1968) have suspected that venom contains a phosphatase which is either specific for or highly preferential to nucleoside diphosphates. Present results support this contention.…”

Presence of O2'-methylpseudouridine in the 18S + 26S ribosomal ribonucleates of wheat embryo

“…Since the true chain lengths of wheat embryo 26S and 18S RNA are not known with certainiy at the present time, this assumed mass ratio may be high. On the other hand, it is known that under certain conditions wheat embryo 26S RNA can undergo a structural alteration with the formation of products the size of 18S RNA (McLennan and Lane, 1968). It has also been shown that wheat embryo NaCl-insoluble RNA contains a heterogeneous population of RNA species migrating on polyacrylamide gels between 26S RNA and 18S RNA and between 18S RNA and satellite RNA (Oakden et al, 1972), and accounting for about 25% of the mass of NaCl-insoluble RNA (Table VII).…”