T4 RNA ligase catalyzes the synthesis of dinucleoside polyphosphates

bT4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 5'-polyphosphates by displacement of the AMP from the E-AMP complex with polyphosphates and nucleoside diphosphates and triphosphates. Displacement of the AMP by tripolyphosphate (P-3) was concentration dependent, as measured by SDS/PAGE. When the enzyme was incubated in the presence of 0.02 mM [alpha-P-32] ATP, synthesis of labeled Ap(4)A was observed: ATP was acting as both donor (K-m, mu M) and acceptor (K-m, mM) of AMP from the enzyme. Whereas, as previously known, ATP or dATP (but not other nucleotides! were able to form the E-AMP complex, the specificity of a compound to be acceptor of AMP from the E-AMP complex was very broad, and with K-m values between 1 and 2 mM. In the presence of a low concentration (0.02 mM) of [alpha-P-32] ATP (enough to form the E-AMP complex, but only marginally enough to form Ap4A) and 4 mM of the indicated nucleotides or P-3, the relative rate of synthesis of the following radioactive (di)nucleotides was observed: Ap(4)X (from XTP, 100); Ap(4)dC (from dGTP, 74); Ap(4)G (from GTP, 49); Ap4dC (from dCTP, 23); Ap(4)C (from CTP, 9); Ap(3)A (from ADP, 5); Ap(4)ddA, (from ddATP, 1); p(4)A (from P-3, 200). The enzyme also synthesized efficiently Ap3A in the presence of 1 mM ATP and 2 mM ADP. The following T4 RNA ligase donors were inhibitors of the synthesis of Ap(4)G: pCp > pAp > pA2'p.