Effects of saturation mutagenesis of the phage SP6 promoter on transcription activity, presented by activity logos

A full set of SP6 promoter variants with all possible single substitutions at positions -17 to +5 was constructed. Transcription activities of these v ariants were individually measured in vivo and in vitro to determine the co ntribution of each base pair to the promoter activity. The in vivo activity was measured indirectly by transcriptional interference of the replication of promoter-bearing plasmids. This activity depends most highly on residue s -11, -9, -8, -7, and +1 (initiation site). All substitutions at -11, -9, -8, and -7 abolished formation of closed complexes, except for A-8C. These residues are involved in base-specific interactions with the polymerase, an d the substitutions exhibit the same strong inhibition in vitro. In contras t, the in vitro activities of some other variants, measured on linearized t emplates, were different from those in vivo. Some variants at -13, -4, and -2, among others, showed exceptionally higher activities in vivo than in vi tro, supporting the possibility that these residues are involved in postbin ding steps, including template melting and bending. The A-3T variant showed much lower activity in vivo than in vitro, but it bound to the polymerase 2-fold more than the consensus sequence and is possibly involved in polymer ase binding. A quantitative hierarchy of all the base pairs is graphically displayed by activity logos, revealing the energetic contribution of each b ase pair to the activity.