Reduced capacity of alternative sigmas to melt promoters ensures stringent promoter recognition.

In bacteria, multiple sigmas direct RNA polymerase to distinct sets of promoters. Housekeeping sigmas direct transcription from thousands of promoters, whereas most alternative sigmas are more selective, recognizing more highly conserved promoter motifs. For sigma(32) and sigma(28), two Escherichia coli Group 3 sigmas, altering a few residues in Region 2.3, the portion of sigma implicated in promoter melting, to those universally conserved in housekeeping sigmas relaxed their stringent promoter requirements and significantly enhanced melting of suboptimal promoters. All Group 3 sigmas and the more divergent Group 4 sigmas have nonconserved amino acids at these positions and rarely transcribe >100 promoters. We suggest that the balance of "melting" and "recognition" functions of sigmas is critical to setting the stringency of promoter recognition. Divergent sigmas may generally use a nonoptimal Region 2.3 to increase promoter stringency, enabling them to mount a focused response to altered conditions.

Control of gene expression at a bacterial leader RNA, the agn43 gene encoding outer membrane protein Ag43 of Escherichia coli.

The family of agn alleles in Escherichia coli pathovars encodes autotransporters that have been implicated in biofilm formation, autoaggregation, and attachment to cells. The alleles all have long leader RNAs preceding the Ag43 translation initiation codon. Here we present an analysis of the agn43 leader RNA from E. coli K-12. We demonstrate the presence of a rho-independent transcription terminator just 28 bp upstream of the main translation start codon and show that it is functional in vitro. Our data indicate that an as-yet-unknown mechanism of antitermination of transcription must be operative in earlier phases of growth. However, as bacterial cell cultures mature, progressively fewer transcripts are able to bypass this terminator. In the K-12 leader sequence, two in-frame translation initiation codons have been identified, one upstream and the other downstream of the transcription terminator. For optimal agn43 expression, both codons need to be present. Translation from the upstream start codon leads to increased downstream agn43 expression. Our findings have revealed two novel modes of regulation of agn43 expression in the leader RNA in addition to the previously well-characterized regulation of phase variation at the agn43 promoter.

Conformational flexibility of sigma(70) in anti-terminator loading.

In promoter DNA, the preferred distance of the -10 and -35 elements for interacting with RNA polymerase-bound sigma(70) is 17 bp. However, the Devi et al. paper in this issue of Molecular Microbiology demonstrates that when the C-terminal domain of sigma(70), including the 3.2 linker, is not attached to the core enzyme, distances between 0 and 3 bp can be accommodated. This attests to the great flexibility of the 3.2 linker. The particularly stable complex with the 2 bp separation may lend itself to structural studies of an early elongation complex containing sigma(70).

The -11A of promoter DNA and two conserved amino acids in the melting region of sigma70 both directly affect the rate limiting step in formation of the stable RNA polymerase-promoter complex, but they do not necessarily interact.

Formation of the stable, strand separated, 'open' complex between RNA polymerase and a promoter involves DNA melting of approximately 14 base pairs. The likely nucleation site is the highly conserved -11A base in the non-template strand of the -10 promoter region. Amino acid residues Y430 and W433 on the sigma70 subunit of the RNA polymerase participate in the strand separation. The roles of -11A and of the Y430 and W433 were addressed by employing synthetic consensus promoters containing base analog and other substitutions at -11 in the non-template strand, and sigma70 variants bearing amino acid substitutions at positions 430 and 433. Substitutions for -11A and for Y430 and W433 in sigma70 have small or no effects on formation of the initial RNA polymerase-promoter complex, but exert their effects on subsequent steps on the way to formation of the open complex. As substitutions for Y430 and W433 also affect open complex formation on promoter DNA lacking the -11A base, it is concluded that these amino acid residues have other (or additional) roles, not involving the -11A. The effects of the substitutions at -11A of the promoter and Y430 and W433 of sigma70 are cumulative.

Substitution of a highly conserved histidine in the Escherichia coli heat shock transcription factor, sigma32, affects promoter utilization in vitro and leads to overexpression of the biofilm-associated flu protein in vivo.

The heat shock sigma factor (sigma(32) in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of sigma(32) is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, sigma(70). This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing sigma(32), implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in sigma(32) impedes recovery from heat shock (exposure to 42 degrees C), and it also leads to overexpression at lower temperatures (30 degrees C) of the Flu protein, which is associated with biofilm formation.

An RNA polymerase mutant deficient in DNA melting facilitates study of activation mechanism: application to an artificial activator of transcription.

Transcription initiation is a major target for the regulation of gene expression in all organisms. Transcription activators can stimulate different steps in the initiation process including the initial binding of RNA polymerase (RNAP) to the promoter and a subsequent promoter-melting step. Typically, kinetic assays are required to determine whether an activator exerts its effect on the initial binding of RNAP or on the promoter-melting step. Here we take advantage of a mutant Escherichia coli RNAP that is deficient in promoter melting to assess the ability of an activator to stabilize the initial binding of RNAP to the promoter. For the well-characterized activator CRP, we show that this RNAP mutant can be used to distinguish between effects on initial binding and promoter melting; these results provide an independent confirmation of the results of kinetic analysis. We then employ the melting-deficient RNAP mutant to demonstrate an effect of an artificial activator of transcription on the initial binding of RNAP. Our findings demonstrate that a melting-deficient RNAP mutant can be used to trap a normally unstable intermediate in transcription initiation, thus providing a novel tool for probing activation mechanism.

Base flipping in open complex formation at bacterial promoters.

In the process of transcription initiation, the bacterial RNA polymerase binds double-stranded (ds) promoter DNA and subsequently effects strand separation of 12 to 14 base pairs (bp), including the start site of transcription, to form the so-called "open complex" (also referred to as RP(o)). This complex is competent to initiate RNA synthesis. Here we will review the role of σ70 and its homologs in the strand separation process, and evidence that strand separation is initiated at the -11A (the A of the non-template strand that is 11 bp upstream from the transcription start site) of the promoter. By using the fluorescent adenine analog, 2-aminopurine, it was demonstrated that the -11A on the non-template strand flips out of the DNA helix and into a hydrophobic pocket where it stacks with tyrosine 430 of σ70. Open complexes are remarkably stable, even though in vivo, and under most experimental conditions in vitro, dsDNA is much more stable than its strand-separated form. Subsequent structural studies of other researchers have confirmed that in the open complex the -11A has flipped into a hydrophobic pocket of σ70. It was also revealed that RPo was stabilized by three additional bases of the non-template strand being flipped out of the helix and into hydrophobic pockets, further preventing re-annealing of the two complementary DNA strands.

Mechanism of bacterial transcription initiation: RNA polymerase - promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis.

Initiation of RNA synthesis from DNA templates by RNA polymerase (RNAP) is a multi-step process, in which initial recognition of promoter DNA by RNAP triggers a series of conformational changes in both RNAP and promoter DNA. The bacterial RNAP functions as a molecular isomerization machine, using binding free energy to remodel the initial recognition complex, placing downstream duplex DNA in the active site cleft and then separating the nontemplate and template strands in the region surrounding the start site of RNA synthesis. In this initial unstable "open" complex the template strand appears correctly positioned in the active site. Subsequently, the nontemplate strand is repositioned and a clamp is assembled on duplex DNA downstream of the open region to form the highly stable open complex, RP(o). The transcription initiation factor, σ(70), plays critical roles in promoter recognition and RP(o) formation as well as in early steps of RNA synthesis.

Evidence for a tyrosine-adenine stacking interaction and for a short-lived open intermediate subsequent to initial binding of Escherichia coli RNA polymerase to promoter DNA.

Bacterial RNA polymerase and a "sigma" transcription factor form an initiation-competent "open" complex at a promoter by disruption of about 14 base pairs. Strand separation is likely initiated at the highly conserved -11 A-T base pair. Amino acids in conserved region 2.3 of the main Escherichia coli sigma factor, sigma(70), are involved in this process, but their roles are unclear. To monitor the fates of particular bases upon addition of RNA polymerase, promoters bearing single substitutions of the fluorescent A-analog 2-aminopurine (2-AP) at -11 and two other positions in promoter DNA were examined. Evidence was obtained for an open intermediate on the pathway to open complex formation, in which these 2-APs are no longer stacked onto their neighboring bases. The tyrosine at residue 430 in region 2.3 of sigma(70) was shown to be involved in quenching the fluorescence of a 2-AP substituted at -11, presumably through a stacking interaction. These data refine the structural model for open complex formation and reveal a novel interaction involved in DNA melting by RNA polymerase.

Threonine 429 of Escherichia coli sigma 70 is a key participant in promoter DNA melting by RNA polymerase.

Initiation of transcription is an important target for regulation of gene expression. In bacteria, the formation of a transcription-competent complex between RNA polymerase and a promoter involves DNA strand separation over a stretch of about 14 base pairs. Aromatic and basic residues in conserved region 2.3 of Escherichia coli sigma(70) had been found to participate in this process, but it is still unclear which amino acid residues initiate it. Here we report an essential role for threonine (T) at position 429 of sigma(70): its substitution by alanine (T429A) results in the largest decrease in open complex formation yet observed for any single substitution in region 2.3. Promoter recognition itself is not affected by T429A substitution, thus providing evidence for a role of T429 in the strand-separation step. Our data are consistent with a model where the T429 would act as a competitor for the hydrogen bonding that stabilizes the highly conserved -11A-T base pairs of the promoter DNA, thus facilitating initiation of strand separation at this particular position in the -10 region. This model suggests an active role for RNA polymerase in disrupting the -11 base pair, rather than just capturing the -11A subsequent to spontaneous unpairing.

Strand opening-deficient Escherichia coli RNA polymerase facilitates investigation of closed complexes with promoter DNA: effects of DNA sequence and temperature.

Formation of the strand-separated, open complex between RNA polymerase and a promoter involves several intermediates, the first being the closed complex in which the DNA is fully base-paired. This normally short lived complex has been difficult to study. We have used a mutant Escherichia coli RNA polymerase, deficient in promoter DNA melting, and variants of the P(R) promoter of bacteriophage lambda to model the closed complex intermediate at physiologically relevant temperatures. Our results indicate that in the closed complex, RNA polymerase recognizes base pairs as double-stranded DNA even in the region that becomes single-stranded in the open complex. Additionally, a particular base pair in the -35 region engages in an important interaction with the RNA polymerase, and a DNase I-hypersensitive site, pronounced in the promoter DNA of the open complex, was not present. The effect of temperature on closed complex formation was found to be small over the temperature range from 15 to 37 degrees C. This suggests that low temperature complexes of wild type RNA polymerase and promoter DNA may adequately model the closed complex.

Mechanistic differences in promoter DNA melting by Thermus aquaticus and Escherichia coli RNA polymerases.

Formation of strand-separated, functional complexes at promoters was compared for RNA polymerases from the mesophile Escherichia coli and the thermophile Thermus aquaticus. The RNA polymerases contained sigma factors that were wild type or bearing homologous alanine substitutions for two aromatic amino acids involved in DNA melting. Substitutions in the sigmaA subunit of T. aquaticus RNA polymerase impair promoter DNA melting equally at temperatures from 25 to 75 degrees C. However, homologous substitutions in sigma70 render E. coli RNA polymerase progressively more melting-defective as the temperature is reduced below 37 degrees C. The effects of the mutations on the mechanism of promoter DNA melting were investigated by studying the interaction of wild type and mutant RNA polymerases with "partial promoters" mimicking promoter DNA where the nucleation of DNA melting had taken place. Because T. aquaticus and E. coli RNA polymerases bound these templates similarly, it was concluded that the different effects of the mutations on the two polymerases are exerted at a step preceding nucleation of DNA melting. A model is presented for how this mechanistic difference between the two RNA polymerase could explain our observations.

Mutational analysis of Escherichia coli heat shock transcription factor sigma 32 reveals similarities with sigma 70 in recognition of the -35 promoter element and differences in promoter DNA melting and -10 recognition.

Upon the exposure of Escherichia coli to high temperature (heat shock), cellular levels of the transcription factor sigma32 rise greatly, resulting in the increased formation of the sigma32 holoenzyme, which is capable of transcription initiation at heat shock promoters. Higher levels of heat shock proteins render the cell better able to cope with the effects of higher temperatures. To conduct structure-function studies on sigma32 in vivo, we have carried out site-directed mutagenesis and employed a previously developed system involving sigma32 expression from one plasmid and a beta-galactosidase reporter gene driven by the sigma32-dependent groE promoter on another in order to monitor the effects of single amino acid substitutions on sigma32 activity. It was found that the recognition of the -35 region involves similar amino acid residues in regions 4.2 of E. coli sigma32 and sigma70. Three conserved amino acids in region 2.3 of sigma32 were found to be only marginally important in determining activity in vivo. Differences between sigma32 and sigma70 in the effects of mutation in region 2.4 on the activities of the two sigma factors are consistent with the pronounced differences between both the amino acid sequences in this region and the recognized promoter DNA sequences.

Sigma 32-dependent promoter activity in vivo: sequence determinants of the groE promoter.

The Escherichia coli transcription factor sigma 32 binds to core RNA polymerase to form the holoenzyme responsible for transcription initiation at heat shock promoters, utilized upon exposure of the cell to higher temperatures. We have developed two ways to assay sigma 32-dependent RNA synthesis in E. coli. The plasmid-borne reporter gene for both is lacZ (beta-galactosidase), driven by the groE promoter. In one application, the cells are exposed to a temperature of 42 degrees C in order to induce accumulation of endogenous sigma 32. The other involves isopropylthiogalactopyranoside (IPTG)-induced synthesis of sigma 32 at 30 degrees C from a gene contained on a second plasmid. The latter employs DnaK(-) cells, which additionally contained a second mutation, inactivating the endogenous sigma 32 gene (Bukau and Walker, EMBO J. 9:4027-4036, 1990). These assays were used to delineate the sequences CTTGA (-37 to -33) and GNCCCCATNT (-18 to -9) as important for sigma 32 promoter activity. At each of the specified base pairs, substitutions were found which reduced promoter activity by greater than 75%. Activity was also dependent upon the number of base pairs separating the two regions.

Interaction of RNA polymerase with forked DNA: evidence for two kinetically significant intermediates on the pathway to the final complex.

RNA polymerase forms competitor-resistant complexes with "forked DNA" templates that are double-stranded from the -35 promoter region through the first base pair of the -10 region, with an additional unpaired A at the 3' end of the nontemplate strand. These types of substrates were introduced recently as model templates for the study of DNA-protein interactions occurring in the early stages of the formation of RNA polymerase-promoter open complexes. We have performed kinetic and equilibrium measurements of interactions of wild-type and mutant RNA polymerases bearing substitutions in the sigma(70) initiation factor, with forked DNA of wild-type and mutant sequence. Our observations reveal that formation of a competitor-resistant complex between RNA polymerase and forked DNA, similar to the formation of open complexes at promoters, is a multistep process, and some of the sequentially formed intermediates along the two pathways share common properties. This work establishes, for the forked template, progression through these intermediates in the absence of downstream DNA and validates the use of forked DNA to determine the effects of changes in promoter or RNA polymerase sequence on the process of open complex formation.

RNA polymerase alters the mobility of an A-residue crucial to polymerase-induced melting of promoter DNA.

Strand separation in promoter DNA induced by Escherichia coli RNA polymerase is likely initiated at a conserved A residue at position -11 of the nontemplate strand. Here we describe the use of fluorescence techniques to study the interaction of RNA polymerase with the -11 base. Forked DNA templates were employed, containing the fluorescent base, 2-aminopurine (2AP), substituted at the -11 position in a single-stranded tail comprising the nucleotides on the nontemplate strand at which base pairing is disrupted in an RNA polymerase-promoter complex. We demonstrate that the presence of 2AP instead of an A at position -11 has no major effect on the accessibility of DNA to DNase I or KMnO(4) in the presence or absence of RNA polymerase, thus justifying the use of templates containing the 2AP substitution in the fluorescence studies. A blue shift of the 2AP fluorescence emission maximum is observed in the presence of RNA polymerase. The results of fluorescence anisotropy decay studies indicate that about 60% of the 2AP residues at -11 are immobilized in an RNA polymerase complex. This value is in good agreement with the fraction of 2AP-substituted templates determined to be in a stable, heparin-resistant complex with RNA polymerase. These results are consistent with the residue at -11 being tightly bound in a hydrophobic pocket of the enzyme.