Role of the Coiled-Coil Tip of Escherichia coli DksA in Promoter Control

Escherichia coli DksA works in conjunction with the small-molecule ppGpp to regulate transcription initiation negatively or positively, depending on the identity of the promoter. DksA is in a class of transcription factors that do not bind directly to DNA such as classical repressors or activators but rather bind in the RNA polymerase (RNAP) secondary channel such as the transcription elongation factors GreA and GreB in E. coli and TFIIS in eukaryotes. We found that substitution for either of two residues in its coiled-coil tip, D74 or A76, eliminates DksA function without affecting its apparent affinity for RNAP. The properties of DksA-Gre factor chimeras indicated that the coiled-coil tip is responsible for the DksA-specific effects on open complex formation. A conservative substitution at position 74, D74E, resulted in a loss of DksA function in both negative and positive control, and an E44D substitution at the analogous position in GreA resulted in a gain of function in both negative and positive control. That a single methylene group has such an extraordinary effect on these transcription factors highlights the critical nature of the identity of coiled-coil tip interactions with RNAP for open complex formation. (C) 2011 Elsevier Ltd. All rights reserved.