DNA-binding properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ(D) proteins

Abstract

σ(D) proteins from Aeribacillus pallidus AC6 and Bacillus subtilis bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the -10 element, and this motif is known to be recognized by σ region 2.4 and critical for promoter activity.

FIG. 1.

The A. pallidus hag regulatory region. The hag gene is predicted to be transcribed as a monocistronic mRNA with a 5′ stem-loop (top). The regulatory region includes a predicted UP element and recognition signals (−35 and −10) for σ^D RNAP. The start site in A. pallidus AC6 was determined by 5′-RACE from RNA isolated from cells grown in LB medium at 60°C with shaking and corresponds to the indicated G (+1). The ribosome-binding site (RBS) and initial coding sequence are indicated. For expression studies (Table 1), promoter-lacZ fusions were generated from the indicated upstream endpoints ({) and either of the two downstream endpoints, designated a and b (}).

FIG. 2.

Promoter recognition by σ^D proteins. The −35 region is recognized by region 4.3 (not shown), and the −10 element is recognized by region 2 and an Arg residue from the amino terminus of region 3 (14). The σ^D_Ap and σ^D_Bs proteins are aligned from the initial portion of region 3 through region 2.3 (note that the direction of the protein sequences is inverted relative to conventional orientation). There are only three amino acid substitutions in this region (identical residues are indicated by dashes) and all residues known to contact DNA are identical. The underlined residues implicated in sequence specific promoter recognition include (σ^D_Ap numbering) R104 in region 3 and R97 and D94 in region 2.4. Residues in region 2.3 corresponding to positions involved in promoter melting in other σ factors are also underlined (15). Fluorescently labeled (6-carboxyfluorescein [6-FAM]) oligonucleotide duplexes were used for fluorescence anisotropy analysis of σ-DNA interactions. The −35 and −10 elements are shaded, and the substituted bases are highlighted. +1 indicates the start site for transcription. WT indicates the wild type. M1 and M2 are mutants with mutations in the core −10 element, and NS represents a nonspecific control DNA.

FIG. 3.

DNA binding by purified σ^D proteins monitored by fluorescence anisotropy (FA). σ^D proteins were added stepwise from 0 to 60 μΜ, and DNA binding was detected as an increase in anisotropy of the probe DNA. Probe DNA was a 50-bp 6-carboxyfluorescein (6-FAM)-labeled hag duplex oligonucleotide (Fig. 2) or a variant altered in the −10 region (M1 [from TCCGATAT to TCCGCGCG] and M2 [from TCCGATAT to TCTAATAT] [substitutions are underlined]). FA experiments (excitation wavelength [λ_ex], 492 nm [slit width = 10 nm]; emission wavelength [λ_em], 492 nm [slit width = 15 nm]) were performed at 40 or 50°C in 100 μl TGED buffer (10 mM Tris-HCl, pH 8.0, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol [DTT]) with 100 nM DNA and 50 mM NaCl. Averages of 6 measurements with an integration time of 9 s were determined.