The functional significance of the conserved motif TX2GR, included in one of the six main regions of amino acid sequence similarity identified in the C-terminal portion of both Escherichia coli DNA polymerase I-like and eukaryotic-type DNA polymerases (Blanco, L., Bernad, A., Blasco, M.A., and Salas, M. (1991) Gene (Amst.) 100, 27-38) has been studied by site-directed mutagenesis in the psi 29 DNA polymerase. A revised multiple alignment of this region, including 61 DNA polymerases belonging to these two superfamilies, is presented. In addition, based on amino acid sequence comparisons and by extrapolation to the crystal structure of T7 RNA polymerase, a similar motif (DX2GR) is predicted to be structurally and functionally equivalent in RNA polymerases, the other class of DNA-dependent polymerases. The severe defect in polymerization displayed by two of the psi 29 DNA polymerase mutants used in this study (T434N and R438I) is interpreted as the consequence of a decreased capacity to stabilize the binding of primer-template DNA structures in a polymerization-competent conformation. These mutants were also severely affected in the formation of terminal protein (TP)-dAMP initiation complex, a reaction in which psi 29 DNA polymerase is able to use the TP as primer.