Analysis of the cooperative thermal unfolding of the td intron of bacteriophage T4

The thermal stability of folded transcripts of the fd intron of bacteriopha ge T4 that carried up to three base substitutions was investigated by tempe rature gradient gel electrophoresis (TGGE) and UV melting. The unfolding of this autocatalytic group I intron is endothermic and entropically driven. Although the effects of mutations in base pairs follow in most cases the ex pected order G-C>A-U>G.U>A.C, the extent of global destabilization varies s trongly according to the helix in which substitutions are located. Effects are more pronounced in the P7 helix which forms, together with the P3 helix , the central pseudoknot of group I introns, The stability of the tertiary fold was also monitored as a function of ionic concentration and of the nat ure of the ion, At low ionic strength, the stabilizing effect of divalent i ons is independent Of the nature of the ion. However, with increasing ionic concentration, stabilization is most pronounced for Mg2+ and less for Mn2 with Ca2+ having intermediate effects. Ammonium ions stabilize folding wit h a similar slope, but at concentrations about 400 times higher than divale nt ions. The apparent enthalpic change associated with the tertiary structu re thermal unfolding increases strongly with increasing concentrations of d ivalent ions,A similar increase is observed with the monovalent ammonium io ns, However, in the presence of NH4+ ions, the apparent enthalpy peaks at 2 .0 M and decreases beyond.