Effect of single amino acid changes in the region of the adenylylation site of T4 RNA ligase

Abstract

Preparation and analysis of a series of mutants of bacteriophage T4 RNA ligase that carry single amino acid changes at or near the site of covalent reaction with ATP (adenylylation) are described. The mutant proteins were constructed by site-directed mutagenesis of the gene for T4 RNA ligase (g63) cloned in M13 vectors, transfer of the mutant genes into a lambda pL-containing expression plasmid, and subsequent expression in Escherichia coli. The results give further evidence that Lys-99 is the adenylylation site and that the residue is also important to step 3 in the RNA ligase mechanism (ligation between acceptor and adenylylated donor). Mutations at Glu-100 or Asp-101 have no effect on adenylylation, but Asp-101 is shown to be crucial to both step 2 (transfer of adenylyl to donor) and step 3.