Several dinucleoside polyphosphates are acceptor substrates in the T4 RNA ligase catalyzed reaction

Several dinucleoside polyphosphates accept cytidine-3',5'-bisphosphate from the adenylylated donor 5'-adenylylated cytidine 5',3'-bisphosphate in the T4 RNA ligase catalyzed reaction. The 5'-adenylylated cytidine 5',3'-bispho sphate synthesized in a first step, from ATP and cytidine-3',5'-bisphosphat e, is used as a substrate to transfer the cytidine-3',5'-bisphosphate resid ue to the 3'-OH group(s) of diguanosine tetraphosphate (Gp(4)G) giving rise to Gp(4)GpCp and pCpGp(4)GpCp in a ratio of approximate to 10 : 1, respect ively. The synthesized Gp(4)GpCp was characterized by treatment with snake venom phosphodiesterase and alkaline phosphatase and analysis (chromatograp hic position and UV spectra) of the reaction products by HPLC. The apparent K-m values measured for Gp(4)G and 5'-adenylylated cytidine 5',3'-bisphosp hate in this reaction were approximate to 4 mm and 0.4 mm, respectively. In the presence of 0.5 mm ATP and 0.5 mm cytidine-3',5'-bisphosphate, the rel ative efficiencies of the following nucleoside(5')oligophospho(5')nucleosid es as acceptors of cytidine-3',5'-bisphosphate from 5'-adenylylated cytidin e 5',3'-bisphosphate are indicated in parentheses: Gp(4)G (100); Gp(5)G (10 1); Ap(4)G (47); Ap(4)A (39). Gp(2)G, Gp(3)G and Xp(4)X were not substrates of the reaction. Dinucleotides containing two guanines and at least four i nner phosphates were the preferred acceptors of cytidine-3',5'-bisphosphate at their 3'-OH group(s).