A basic/hydrophobic cleft of the T4 activator MotA interacts with the C‐terminus of E. coli σ70 to activate middle gene transcription

Transcriptional activation often employs a direct interaction between an activator and RNA polymerase. For activation of its middle genes, bacteriophage T4 appropriates Escherichia coli RNA polymerase through the action of two phage-encoded proteins, MotA and AsiA. Alone, AsiA inhibits transcription from a large class of host promoters by structurally remodelling region 4 of sigma(70), the primary specificity subunit of E. coli RNA polymerase. MotA interacts both with sigma(70) region 4 and with a DNA element present in T4 middle promoters. AsiA-induced remodelling is proposed to make the far C-terminus of sigma(70) region 4 accessible for MotA binding. Here, NMR chemical shift analysis indicates that MotA uses a 'basic/hydrophobic' cleft to interact with the C-terminus of AsiA-remodelled sigma(70), but MotA does not interact with AsiA itself. Mutations within this cleft, at residues K3, K28 and Q76, both impair the interaction of MotA with sigma(70) region 4 and MotA-dependent activation. Furthermore, mutations at these residues greatly decrease phage viability. Most previously described activators that target sigma(70) directly use acidic residues to engage a basic surface of region 4. Our work supports accumulated evidence indicating that 'sigma appropriation' by MotA and AsiA uses a fundamentally different mechanism to activate transcription.