T4 RNA ligase: New structural studies on an unusual but useful enzyme.

ABSTRACT

Gene 63 from bacteriophage T4 encodes a single polypeptide with two independent enzyme activities, RNA ligase and tail fibre attachment. The DNA sequence of gene 63 has been determined and the gene cloned in an expression plasmid (pDR540) that contains the inducible tac promoter. Escherichia coli cells containing the plasmid (KR54) produce about 5–10 % of their soluble protein as RNA ligase. A convenient isolation procedure for the enzyme is described from KR54 cells and the isolated product is indistinguishable from that obtainable from T4-infected Escherichia coli. The enzyme reacts reversibly with ATP in the presence of Mg2+ to give a covalent AMP-enzyme adduct. It is shown by FAB mass spectrometric analysis of chymotryptic fragments of the adenylylated enzyme that the AMP is bound covalently to lysine residue 99. Methods of in vitro mutagenesis are described for gene 63 cloned in a bacteriophage M13 vector. By deletion mutagenesis it was shown that the C-terminal 20 % of the protein is not crucial for the RLi activity but the TFA activity, as measured by a complementation assay, is reduced. A method is described for the introduction of point mutations in gene 63 by use of AMV reverse transcriptase for error-directed repair polymerisation in gapped DNA heteroduplexes. In addition, a synthetic oligonucleotide mismatched at the 3’ end was used as a primer for reverse transcriptase catalysed repair polymerisation to force a single base change in the codon for Lys-99 to give the codon for Asn. The mutant protein has no detectable RNA ligase activity but retains tail fibre attachment activity.