Joining of ribooligonucleotides with T4 RNA ligase and identification of the oligonucleotide-adenylate intermediate.
Open Access
- 1 June 1976
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 3 (6) , 1613-1624
- https://doi.org/10.1093/nar/3.6.1613
Abstract
T4 RNA ligase was found to join A-A-A-A-A-A and 32pU-U-U-U in the presence of ATP as cofactor. In this reaction the pyrophosphate of *pU-U-U-U and pA was isolated by chromatography on a RPC-5 column, besides the joined product and the starting materials. This pyrophosphate was shown to be an intermediate in the joining reaction because of the fact that coupling with A-A-A-A-A-A to give the decanucleotide could be performed in the absence of ATP. The structure of the oligonucleotide-adenylate was determined by enzymatic digestion with base-nonspecific nuclease and venom phosphodiesterase. Further evidence for the proposed structure was obtained by isolation of the intermediate obtained by using *pU-U-U-U and [α-32p]ATP. This pyrophosphate gave *pA and *pU by treatment with venom phosphodiesterase. Several other joining reactions between various purine- and pyrimidine ribooligonucleotides to 5′-phosphorylated ribooligonucleotides are discussed.Keywords
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