Addition of oligonucleotides to the 5'-terminus of DNA by T4 RNA ligase.
Bacteriophage T4-induced RNA ligase catalyzes the controlled template-independent addition of RNA to the 5'-phosphoryl end of large DNA molecules. Restriction enzyme-generated fragments of Co1E1 DNA with completely basepaired or cohesive ends and linear single-stranded øX174 viral DNA were all good substrates. DNA molecules from 10 to 6000 nucleotides long were quantitatively joined in an hour to a number of different RNA homopolymers. The most efficient of these was A(pA)5; I(pI)5 and C(pC)5 were also utilized while U(pU)5 was not. The optimum ribohomopolymer length was six nucleotides. Joining of ribohomopolymers between 10 and 20 nucleotides long occurred at approximately 1/2 the maximal rate and a trimer was the shortest substrate. Thus RNA ligase provides a method for generating extensions of predetermined length and base composition at the 5'-end of large DNA molecules that complements the available procedures for extending the 3'-hydroxyl terminus of DNA.
D003090:Coliphages / Q000201:enzymology*; D004247:DNA; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D011118:Polynucleotide Ligases / Q000378:metabolism*; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D013379:Substrate Specificity
D009841:Oligonucleotides; D009843:Oligoribonucleotides; D004247:DNA; D004262:DNA Restriction Enzymes; D011118:Polynucleotide Ligases; D011130:RNA Ligase (ATP)