RNA Ligase Activity in Phage-Infected Bacteria and Animal Cells

Abstract

An RNA ligase which is dependent on ATP and Mg2+ for maximal activity with 5′-32P-labeled poly(I) · poly(C) as substrate has been purified from Escherichia coli infected with phage T4. Ligase activity was demonstrated by the transfer of alkaline phosphatase sensitive 5′-32P termini into an alkaline phosphatase resistant form. The formation of phosphodiester bond was verified by hydrolysis of the reaction product and determining the transfer of 32P from the 5′ position to the 2′(3′) position by electrophoresis. An increase in sedimentation rate of poly(I) · poly(C) was also observed after reaction with RNA ligase. The enzyme also showed an ATP-PPi exchange activity. The activity on the synthetic polyribonucleotides poly(A), poly(U), poly(A) · poly(U) and poly(I) · poly(C) was examined and an increased efficiency of the substrate was obtained when [5′-32P]poly(U) formed a complex with poly(A). [5′-32P]poly(A) was a good substrate both with and without the complementary strand. With 5′-32P-labeled poly(I) · poly(C) as substrate RNA-ligase activity was detected in mammalian cells such as KB, HeLa and L-cells. The activity seems to increase after infection with encephalomyelocarditis (EMC) virus or poliovirus, but not after infection with adenovirus type 2. The RNA ligase activity was confined to the cytoplasmic fraction of the cells.