Respective role of each of the purine N-6 amino groups of 5'-O-triphosphoryladenylyl(2'----5')adenylyl(2----5')adenosine in binding to and activation of RNase L.
We have synthesized a series of 2-5A (ppp5'-A2'p5'A2'p5'A) analogs in which each adenosine residue has been sequentially replaced by inosine: viz., ppp5'I2'p5'A2'p5'A, ppp5'A2'p5'I2'p5'A, and ppp5'A2'p5'A2'p5'I. These transformations enabled us to delineate the role of each of the three purine N-6 amino groups of 2-5A in determining oligonucleotide binding to and activation of the 2-5A-dependent endoribonuclease, RNase L. With the RNase L activity of both mouse L cells and human Daudi lymphoblastoid cells, we found that the N-6 amino group of the first adenosine nucleotide residue (from the 5'-terminus) is of crucial importance in determining binding to the endonuclease; however, removal of the N-6 amino moieties of the second or third adenosine nucleotide residues resulted in only a minimal decrease in binding to the endonuclease. On the other hand, conversion of the third adenosine residue to inosine effected a dramatic (10,000-fold compared to 2-5A) loss in ability to activate the nuclease; however, execution of the same N-6 amino group conversion at either the first or second adenosine residue did not cause a major change in nuclease activation ability when the accompanying decreased endonuclease binding was considered. These results clearly demonstrate that the N-6 amino group of the first adenosine residue of 2-5A is critical in RNase L binding whereas the N-6 amino function of the third adenosine residue of 2-5A is crucial for the activation of RNase L.
Imai
J
J
Lesiak
K
K
Torrence
P F
PF
D000227:Adenine Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D004686:Encephalomyocarditis virus / Q000235:genetics; D004722:Endoribonucleases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007288:Inosine; D051379:Mice; D009841:Oligonucleotides / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000378:metabolism* / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D012335:RNA, Ribosomal / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D013329:Structure-Activity Relationship
D000227:Adenine Nucleotides; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D012335:RNA, Ribosomal; D012367:RNA, Viral; D007288:Inosine; C023505:2',5'-oligoadenylate; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease