Joining of ribooligonucleotides with T4 RNA ligase and identification of the oligonucleotide-adenylate intermediate.
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. Futher evidence for the proposed structure was obtained by isolation of the intermediate obtained  by using pU-U-U-U and [alpha-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.
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D003090:Coliphages / Q000201:enzymology*; D011756:Diphosphates / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases; D011118:Polynucleotide Ligases / Q000378:metabolism*; D012260:Ribonucleases; D014542:Uridine Monophosphate / Q000378:metabolism
D011756:Diphosphates; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D014542:Uridine Monophosphate; D012260:Ribonucleases; D010727:Phosphoric Diester Hydrolases; D011118:Polynucleotide Ligases