Studies on Ribonucleic Acid Ligase

Abstract

Abstract RNA ligase, isolated from bacteriophage T4-infected Escherichia coli, catalyzes the conversion of synthetic [5'-32P]polyribonucleotides to a form resistant to alkaline phosphatase. The reaction requires ATP and leads to the formation of AMP and PPi. The purification procedure for T4-induced RNA ligase has been modified to yield stable enzyme preparations. The influence of secondary structure on the RNA ligase reaction has been investigated with these fractions. The conversion of synthetic polynucleotides to complex secondary structures was either without effect or inhibitory in the RNA ligase system. Purified RNA ligase was shown to catalyze an exchange reaction between ATP and PPi, but not between ATP and AMP. The ATP-PPi exchange reaction occurred in the absence of a polyribonucleotide substrate. The formation of an RNA ligase-adenylate complex was demonstrated by gel filtration, polyacrylamide gel electrophoresis, and ultracentrifugation in a glycerol gradient. The complex was dissociated by 5'-P-poly(A) to yield AMP while ATP was generated in the presence of PPi. These findings suggest that an enzyme-AMP intermediate occurs during the course of the RNA ligase reaction. Evidence is presented for the occurrence of an RNA ligase activity in eukaryotic cells.