The class I aminoacyl-tRNA synthetases are defined by an N-terminal nucleotide binding fold that contains the active site for adenylate synthesis. Insertions and additions of idiosyncratic RNA binding elements that facilitate docking of the L-shaped tRNA structure are superimposed onto this basic fold. These RNA binding elements are imagined to have been acquired during the evolution and development of the modern genetic code. The monomeric Escherichia coli isoleucyl-tRNA synthetase has a zinc-containing peptide at its C terminus. Removal of the zinc-containing peptide was shown previously to create a shortened enzyme with activity for adenylate synthesis but no detectable binding to tRNA(Ile). We show here that the isolated zinc-containing peptide binds to tRNA with relatively low affinity. This binding is not tRNA-specific but shows a strict requirement for zinc. In contrast, the zinc-containing peptide conferred specific and high-affinity binding when combined with the shortened enzyme. Thus, when combined with another protein, a nonspecific tRNA binding peptide is essential for formation of a high-affinity and specific tRNA binding site. These results demonstrate the feasibility of the idea that noncovalent complexes of general RNA-binding peptides with a domain for adenylate synthesis were precursors to modern tRNA synthetases. In addition, the results offer the first direct evidence of a role for zinc in the tRNA-binding activity of one of these peptide elements.