Previous studies have led to a model in which the promoter-specific recognition of prokaryotic transcription initiation factor, sigma (σ), is core dependent. Most σ functions were studied on the basis of this tenet. Here, we provide in vitro evidence demonstrating that the intact Bacillus subtilis primary sigma, σA, by itself, is able to interact specifically with promoter deoxyribonucleic acid (DNA), albeit with low sequence selectivity. The core-independent promoter-specific interaction of the σA is −10 specific. However, the promoter −10 specific interaction is unable to allow the σA to discern the optimal promoter spacing. To fulfill this goal, the σA requires assistance from core RNA polymerase (RNAP). The ability of σ, by itself, to interact specifically with promoter might introduce a critical new dimension of study in prokaryotic σ function.

Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant containing double-amino-acid substitutions (I198A and I202A) on the hydrophobic face of the promoter -10 binding helix of sigma(A) factor. We have analyzed the structural and functional properties of this mutant sigma(A) factor both in vivo and in vitro. Our data revealed that the Ts sigma(A) factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts sigma(A) resulted in a very low core-binding activity of the Ts sigma(A) factor and a markedly reduced sigma(A)-RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts sigma(A) is the main trigger for the temperature sensitivity of B. subtilis DB1005. Partial proteolysis, tryptophan fluorescence and 1-anilinonaphthalene-8-sulfonate-binding analyses revealed that the hydrophobic face of the promoter -10 binding helix and also the hydrophobic core region of the Ts sigma(A) factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts sigma(A) and allows the formation of multimeric Ts sigma(A). Our results also indicate that Ile-198 and Ile-202 on the hydrophobic face of the promoter -10 binding helix are essential to ensure the correct folding and stabilization of the functional structure of sigma(A) factor.

Bacillus subtilis DB1005 is a temperature‐sensitive (Ts) sigA mutant containing double‐amino‐acid substitutions (I198A and I202A) on the hydrophobic face of the promoter −10 binding helix of σA factor. We have analyzed the structural and functional properties of this mutant σA factor both in vivo and in vitro. Our data revealed that the Ts σA factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts σA resulted in a very low core‐binding activity of the Ts σA factor and a markedly reduced σA‐RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts σA is the main trigger for the temperature sensitivity of B. subtilis DB1005. Partial proteolysis, tryptophan fluorescence and 1‐anilinonaphthalene‐8‐sulfonate–binding analyses revealed that the hydrophobic face of the promoter −10 binding helix and also the hydrophobic core region of the Ts σA factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts σA and allows the formation of multimeric Ts σA. Our results also indicate that Ile‐198 and Ile‐202 on the hydrophobic face of the promoter −10 binding helix are essential to ensure the correct folding and stabilization of the functional structure of σA factor. Proteins 2000;40:613–622. © 2000 Wiley‐Liss, Inc.

Bacillus subtilis r D is an alternative r factor that possesses a core-independent promoter À10 element binding specificity despite the lack of a distinct footprint on its cognate promoter. We wished to determine whether this property is common to alternative r factors. To this end, we overexpressed B. subtilis r B in Escherichia coli and analyzed its DNA binding ability by electrophoretic mobility shift assay and DNase I footprinting. The major complex formed by r B and its cognate promoter DNA is heparin-sensitive. However, in contrast to the À10 element binding specificity observed for B. subtilis r D , the promoter binding of r B is specific for the À35 element. These and other results clearly demonstrate that alternative r factors possess different promoter-binding characteristics, and make coreindependent contributions to recognition of their cognate promoters.

Sigma (σ) factors are bacterial transcription initiation factors that direct transcription at cognate promoters. The promoters recognized by primary σ are composed of -10 and -35 consensus elements separated by a spacer of 17±1 bp for optimal activity. However, how the optimal promoter spacing is sensed by the primary σ remains unclear. In the present study, we examined this issue using a transcriptionally active Bacillus subtilis N-terminally truncated σA (SND100-σA). The results of the present study demonstrate that SND100-σA binds specifically to both the -10 and -35 elements of the trnS spacing variants, of which the spacer lengths range from 14 to 21 bp, indicating that simultaneous and specific recognition of promoter -10 and -35 elements is insufficient for primary σ to discern the optimal promoter spacing. Moreover, shortening in length of the flexible linker between the two promoter DNA-binding domains of σA also does not enable SND100-σA to sense the optimal promoter spacing. Efficient recognition of optimal promoter spacing by SND100-σA requires core RNAP (RNA polymerase) which reduces the flexibility of simultaneous and specific binding of SND100-σA to both promoter -10 and -35 elements. Thus the discrimination of optimal promoter spacing by σ is core-dependent.