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Fig. 4 | BMC Microbiology

Fig. 4

From: Crystal structure and DNA binding activity of a PadR family transcription regulator from hypervirulent Clostridium difficile R20291

Fig. 4

EMSAs of cdPadR1 binding the cdpadR1 promoter (P cdpadR1 ) and smaller regions containing predicted binding boxes. a P cdpadR1 fragments that were bound by cdPadR1 are illustrated in green above and those that were not bound are illustrated in gray below the P cdpadR1 sequence. The predicted -10 and -35 sites are indicated in blue boxes above the sequence. b Final P cdpadR1 (Pr27, 100 bp) concentration in the reaction was 0.05 μM. cdPadR1 was 5, 10, 20, and 40-fold excess over DNA. c-d Final Pr32 (64 bp) and Pr31 (61 bp) concentration in the reactions were 0.1 μM. cdPadR1 was 5, 10, 20, and 40-fold excess over DNA. e-f The 21 bp P cdpadR1 fragment (Pr68) contains predicted binding boxes 1 and 2 (orange arrows) separated by 11 bp and contains a 1 bp overhang on both the 5′ and 3′ ends. The 30 bp P cdpadR1 fragment (Pr122) contains predicted binding boxes 3 and 4 (orange arrows) and contains 5 bp overhangs on both the 5′ and 3′ ends. Final Pr68 (21 bp) and Pr122 (30 bp) concentration in the reactions were 0.25 μM. cdPadR1 was 2, 4, 6, and 8-fold excess over DNA. g Final dsDNA concentration varied depending on the size of the fragment; the + lane contains 10-fold excess cdPadR1 over DNA. For EMSA gels B-F the minus (-) lane contains DNA and no cdPadR1. Shifted DNA-protein complexes are annotated with a black arrow and unbound DNA migration is marked with a red arrow

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