In order to demonstrate the essential role of the evolutionarily conserved hairpin structure in the regulation of Pol b expression, we carried out in vitro mutagenesis to disrupt the M2 hairpin element. Mutagenic primers were designed based on an MFOLD (14) prediction of the potential effect of nucleotide change on hairpin structure ( Figure 3 ). Substitution of three C-residues for three G-residues in the stem-forming region (positions 67-69) changed the predicted free energy increment (dG) from À14.1 kcal/mol for the intact structure to À3.6 kcal/mol for the mutant. The mutated sequence (Hmut) was used in luciferase reporter assays and served as a template for in vitro transcription to generate mRNA for subsequent crosslink and filter-binding analysis.