The class I methionyl tRNA synthetase has a conserved N-terminal nucleotide binding fold which contains the active site, and a largely non-conserved C-terminal anticodon binding domain. At the C-terminal end of the anticodon binding domain is a peptide which curls back into the N-terminal nucleotide binding fold near the active site. We showed that a mutation in this peptide disrupts aminoacylation and binding of a 7 base pair microhelix substrate based on the acceptor stem of tRNA(fMet). The novel technique of affinity coelectrophoresis was applied to this system for the first time to determine dissociation constants of wild-type and mutant MetRS for small RNA substrates. A description and evaluation of this technique for measuring weak protein-nucleic acid interactions is presented here, in the context of the methionyl tRNA synthetase system.