Fig. 2

Structural and functional analysis of the DNA-binding model of the Stl dimer. a Model of dimeric Stl bound to the palindromic SaPIbov1 repression site. The BovI-Stl^N-ter dimer is presented in cartoon representation with one protomer coloured as in Fig. 1b and the other with lighter tones. DNA is shown in grey sticks and white surface with the TATCTC palindromic sequences highlighted in orange. Binding of the dimer is based on the superimposition of individual BovI-Stl^N-ter protomers with the C.Esp1396I complex and its palindromic DNA operator (PDB:3CLC⁴¹; Supplementary Fig. 2b). Structural elements of the HTH DNA-binding domain and the connector helix are labelled. b Close view of the modelled HTH-DNA interaction. Stl DNA interaction residues are highlighted in stick, coloured by atom type and labelled. The sulfate ion that is presented in the BovI-Stl^N-ter structure is also shown in stick and labelled, and superimposes on a DNA backbone phosphate. The HTH α-helices are labelled. c Unregulated str transcription due to SaPIbov1 Stl repressor DNA- binding domain mutations. Top left: schematic of a blaZ transcriptional fusion in pJP2085. β-Lactamase assays were performed with RN4220 containing the pJP2085 SaPIbov1 Stl^WT or derivatives, with data from three independent experiments. Error bars represent SD. ANOVA with Tukey’s multiple comparisons test compared mean differences between the Stl^WT control and the mutants. Significant adjusted p values relative to the WT were: Stl^R22A < 0.0001^****; Stl^Q29A < 0.0001^****; Stl^S44A < 0.0001^****; Stl^N45A < 0.0001^****; Stl^E47A < 0.0001^****; Stl^N48A < 0.0001^****; Stl^R51A < 0.0001^****; Stl^R74A < 0.0001^****. d In vitro evaluation of mutations in the Stl DNA-binding domain. EMSA gel results of Stl^WT and mutants in the residues predicted to interact with DNA in the model of panel b. In the presence of Stl^WT the band corresponding to the stl-str DNA operator region, labelled as free DNA, disappears and two new bands corresponding to Stl-DNA complexes are observed (labelled as complex I and II; see ref. ²¹). For the Stl mutants the bands corresponding to Stl-DNA complexes almost completely disappear while most of the DNA appears at the lower position of the gel as free DNA. One representative experiment of at least three independent replicates is shown. Source data are provided as a Source Data file