Figure 5.

The binding of STM1697 to FlhD₄C₂ restrains RNAP recruitment. (A) Sequence analysis of the upstream region of the class II flagellar gene fliA. The FlhD₄C₂ binding region is highlighted in purple, and the −10 like region of the promoter that serves as a σ⁷⁰ RNA polymerase binding site is colored green. The transcriptional start site is marked in red. The endpoints of the 269 bp DNA used for the following experiments is also shown. (B) Upper: FlhD₄C₂ complex interacts with target DNA through the Zn-cys cluster and the positive-charge-enriched region of the FlhC dimer. After binding to the −30 to −70 region of the target gene, the FlhD₄C₂ complex recruits RNA polymerase to form a transcription initiation complex, then the transcription of class 2 flagellar genes is turned on. Lower: the STM1697 molecules bind to two peripheral FlhD subunits to form three-protein complexes with FlhD₄C₂. The incorporation of STM1697 prevents RNA polymerase from binding the promoter. As a result, the subsequent expression of flagellar genes is repressed. (C–E) Native gel results preformed with different ratios of protein mixture. Twenty picomoles of FlhD₄C₂, 10 pmol of DNA and 40 pmol of RNAP were used in the reaction systems. Different amounts of YdiV, STM1344 or STM1697 were added according to the indicated ratio to FlhD₄C₂. The upper and lower pictures show the same gel stained with Coomassie brilliant blue and Gel red, respectively. Band A is the complex of RNAP, FlhD₄C₂ and DNA, and band B contains FlhD₄C₂ and DNA. When YdiV, STM1344 or STM1697 were added, band A disappeared.