Structural and functional properties of a Bacillus subtilis temperature‐sensitive σA factor

Abstract

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.

Properties of Bacillus subtilis σ A Factors with Region 1.1 and the Conserved Arg-103 at the N Terminus of Region 1.2 Deleted

“…The dispensability of this region for B. subtilis A to function (Fig. 2, 3, 5, and 6) implies that it can be independent and separated from other domains of further support also comes from our previous study, in which Arg-103 was shown to be more accessible to trypsin digestion (34). It has been proposed that the acidic region 1.1 of Taq A localizes directly inside the main channel and interacts with the basic surface of the channel wall of the RNA polymerase core enzyme (23).…”

The Importance of Region 2.1 in Sustaining the Functional Structure of the Bacillus subtilis AFactor

“…2) is quite different from that observed for the Ts sigA mutant (B. subtilis DB1005) with Ile-198, 202-Ala substitutions on the hydrophobic face of the promoter -10 binding helix of cr*, of which the growth potential is comparable to that of the Wt at 37°C (12). We speculated that the replacement with alanine of the bulky hydrophobic amino acid on the second helix of region 2.1 had affected the structure of cr* differently from the reported effect of the Ile-to-Ala substitution in region 2.4 of cr* (13).…”

“…3). With G3b promoter as DNA template, the relative activity of RNA polymerase containing the Wt-, L145A-, I149A-, or Y^A-cr* was in the ratio of L145A, I149A, and Y153A Cause Structural Instability of a^ Even at 37°C-The decreased transcription activity of a mutant a factor may be attributed to structural instability (38)(39)(40), aggregation of folding intermediate (13), or low core-binding activity of the mutant cr factor (17,(24)(25). It could also be a result of low promoter-binding activity, change in promoter specificity (5,(9)(10)(11), blocking of transcription initiation (41), or defective open complex formation of the mutant cr-containing RNA polymerase (42,43).…”

“…monomeric form of the Ts cr*, which possesses a looser conThe CT factor confers the specificity of promoter recognition formation than the wild-type (Wt) monomer, was scarcely and the transcription of specific genes. Amino acid se-observed in vitro (13). It is believed that the aggregative quence alignment of various cr factors in the cr 70 family has tendency and looser conformation of the Ts cr* are responsirevealed four regions of homology (1,2).…”

“…Region structure of a partial peptide fragment of the E. coli cr 70 2.4, which is supposed to recognize the promoter -10 DNA, (16), it is assumed that the loose conformation of the monoforms an amphiphilic a-helix with three isoleucines located meric Ts cr* and the tendency to aggregation of the multimat four-residue intervals (12). The three isoleucines on the eric Ts cr* are attributable, at least in part, to the loss of hydrophobic face of the amphiphilic a-helix are critical to contact between the conserved isoleucine residues (1194, ensure a proper folding and to sustain the functional struc-1198, and 1202) on the hydrophobic face of the promoterture of the Bacillus subtilis cr* factor (13).…”