Genetic and physiological studies of Bacillus subtilis sigma A mutants defective in promoter melting

Abstract

The Bacills subtilis sigA gene encodes the primary cr factor of RNA polymerase and is essential for cell growth. We have mutated conserved region 2.3 of the &-protein to substitute each of seven aromatic amino acids with alanine. Several of these aromatic amino acids are proposed to form a melting motif which facilitates the strand separation step of initiation. Holoenzymes containing mutant cf factors recognize promoters, but some are defective for DNA melting in vitro. We have studied the ability of each mutant a factor to support cell growth by gene replacement and complementation. The two region 2.3 mutants least impaired in promoter melting in vitro (Y180A and Y184A) support cell growth in single copy, although the Y184A allele imparts a slow-growth phenotype at low temperatures. A strain expressing only the Y189A variant of the c protein, known to be defective in DNA melting in vitro, grows very slowly and is altered in its pattern of protein synthesis. Only the wild-type and Y180A eA proteins eficiently complement a temperature-sensitive allele of sigA. Overexpression of three of the eA proteins defective for promoter melting in vitro (Y189A, W192A, and W193A) leads to a decrease in RNA synthesis and cell death. These results indicate that mutations which specifically impair DNA melting in vitro also impair (v function in vivo and therefore support the hypothesis that r plays an essential role in both DNA melting and promoter recognition.The a subunit was originally discovered as a specificity factor during biochemical studies of transcription initiation (3). The subsequent demonstration that a factor can be replaced during phage growth (8, 30) and Bacillus subtilis growth transitions (12, 16) helped establish uf as a primary determinant of promoter selectivity, a view supported by recent genetic (9, 27, 34) and biochemical (7) studies.Genetic analyses suggest that most bacteria have a single essential ar factor responsible for the synthesis of stable RNAs and the bulk of mRNA. For example, growth of cells containing a temperature-sensitive allele of Escherichia coli a0 at the nonpermissive temperature results in an approximately 10-fold drop in the rate of total RNA synthesis (11, 13). In contrast, mutants defective in many of the alternative a factors are viable, though often conditionally lethal, and bulk RNA synthesis rates are not grossly altered. The division of a factors into essential, primary or factors and dispensable, alternative or factors is also supported by sequence-based phylogenetic comparisons: all of the primary r factors form a closely related cluster (14,22).Although the role of a factors in determining promoter recognition is now well established (7,9,27,34), the possibility that a contributes to the promoter-melting step in transcription initiation has been controversial. A melting function for a was first postulated to explain the requirement for a during transcription initiation on intact duplex templates but not on DNA containing single-stranded regions (15). In addition, direct ...

A Saccharomyces cerevisiae Mitochondrial Transcription Factor, sc-mtTFB, Shares Features with Sigma Factors but Is Functionally Distinct

“…One such search identified regions within sc-mtTFB that exhibit some similarity to the conserved regions 2 and 3 of sigma factors (13). These regions of sigma factors have been proposed to be involved in recognition of the bacterial promoter (6,8,10,14,30,34,35,39), DNA unwinding (1,15,26,36), and interactions between the sigma factors and the core RNA polymerase (17,38). The significance of these matches has been questioned (19) and is difficult to evaluate for several reasons.…”

“…Specific subregions have been defined within the conserved region 2 of bacterial sigma factors (subregions 2.1 to 2.4). Subregion 2.4 is involved in specific recognition of the Ϫ10 DNA element of the bacterial promoter (6,8,10,14,30,34,35,39), and subregion 2.3 has been implicated in facilitating the unwinding of DNA during transcription initiation (1,15,26,36). Deletions in subregion 2.1 (17) or region 3 (38) of sigma factors apparently reduce the affinity of sigma factors for the core RNA polymerase, suggesting that these regions are necessary for this interaction.…”

Open complex formation by Escherichia coli RNA polymerase: the mechanism of polymerase‐induced strand separation of double helical DNA

“…This melting defect can be overcome by template supercoiling and by raising the reaction temperature (Aiyar et ai, 1994b;Juang and Helmann, 1994). Consistent with a defect in melting, several of these a factors exert a /rans-dominant lethal effect in vivo: their expression leads to a decline in RNA synthesis and cell death (Rong and Helmann, 1994). Since an adjacent grouping of amino acids (region 2.4) participates in -10 recognition (Daniels etai.…”

The essential activities of the bacterial sigma factor

“…1e) (Feklistov and Darst 2011;Zhang et al 2012). An understanding of the importance of aromatic region 2.3 amino acids for promoter melting and of nucleotide -11A for -10 recognition and nucleation of strand-opening first emerged from early studies with B. subtilis RNAP (Juang and Helmann 1994;Rong and Helmann 1994;Qiu and Helmann 1999) and E. coli RNAP (Panaghie et al 2000). In the A -11 binding pocket, a tyrosine (Y253, Fig.…”