Ada protein-RNA polymerase σ subunit interaction and α subunit-promoter DNA interaction are necessary at different steps in transcription initiation at the Escherichia coli ada and aidB promoters

The methylated form of the Ada protein ((me)Ada) binds the ada and aidB promoters between 60 and 40 base pairs upstream from the transcription start and activates transcription of the Escherichia coli ada and aidB genes. This region is also a binding site for the a subunit of RNA polymerase and resembles the rrnB P1 UP element in A/T content and location relative to the core promoter, In this report, we show that deletion of the C-terminal domain of the a subunit severely decreases (me)Ada-independent binding of RNA polymerase to ada and aidB, affecting transcription initiation at these promoters, We provide evidence that (me)Ada activates transcription by direct interaction with the C-terminal domain of RNA polymerase sigma(70) subunit (amino acids 574-613), Several negatively charged residues in the sigma(70) C-terminal domain are important for transcription activation by (me)Ada; in particular, a glutamic acid to valine substitution at position 575 has a dramatic effect on (me)Ada-dependent transcription. Based on these observations, we propose that the role of the a subunit at ada and aidB is to allow initial binding of RNA polymerase to the promoters. However, transcription initiation is dependent on (me)Ada-sigma(70) interaction.