Dissecting the functional program of Escherichia coli promoters: the combined mode of action of Lac repressor and AraC activator.

The mode of action of regulated promoters is largely determined by kinetic parameters which govern the interaction between promoters and proteins involved in induction and repression of transcription. To gain insight into the interplay between positively and negatively acting transcriptional regulators, in this case AraC and LacR, we have generated a panel of promoter sequences derived from P(lac), the promoter of the Escherichia coli lac operon. The function of these promoters is limited at different steps and to various extents within the pathway of RNA polymerase (RNAP)/promoter interaction. Moreover, in all promoters the cAMP receptor protein binding site was replaced by the binding motif of AraC to prevent pleiotropic effects in vivo upon activation. Analyzing the activation of these promoters by AraC in vivo under conditions of repression by LacR and derepression yielded a three step model of transcription initiation which reveals mechanisms of AraC and LacR action. Our data show three distinct rate limiting steps at which AraC can exert its function. In general, the activator accelerates the formation of the first stable complex between RNAP and promoter. At most promoter sequences, however, its main impact is on the conversion of the closed to the open complex. However, AraC is also capable of eliminating limitations at steps following open complex formation.

In vitro evolution of molecular cooperation in CATCH, a cooperatively coupled amplification system.

BACKGROUND: One of the key issues in the investigation of evolution is how complex systems evolved from simple chemical replicators. Theoretical work proposed several models in which complex replicating systems are kinetically stabilized. The development of powerful isothermal amplification technique allows complex nucleic acid based evolving in vitro systems to be set up, which may then serve to verify experimentally current theories of evolution. Recently such a system based on the 3SR (self-sustained sequence replication) reaction has been established to investigate the evolution of cooperation: the trans-cooperatively coupled CATCH (cooperative amplification by cross hybridization). RESULTS: Over four rounds of serial transfer, the cooperatively coupled two species CATCH system evolved into a more complex cooperative four species system, which then was overgrown by CATCH-derived RNA-Z-like hairpin species. In contrast to the classical RNA-Z species, these molecules have complementary loop sequences and self-amplify using a dual mechanism that includes concentration-dependent phases of noncooperative and cooperative amplification. CONCLUSIONS: The evolution of a cooperative system, under conditions that were alternately unfavorable and favorable for cooperative amplification, led to a system showing facultative cooperation. This principle of facultative cooperation preserves the complexity of the system investigated and could have general implications for the evolution and stabilization of cooperation under oscillating reaction conditions.

Cooperative amplification of templates by cross-hybridization (CATCH).

In vitro amplification systems not only serve as a tool for the processing of DNA, but have also provided important model systems for the investigation of fundamental issues in evolutionary optimization. In this work we present a coupled amplification system based on the self-sustained sequence replication (3SR), also known as nucleic acid sequence-based amplification (NASBA), which allows the experimental investigation of evolving molecular cooperation. The 3SR reaction is an isothermal method of nucleic acid amplification and an alternative to PCR. A target nucleic acid sequence can be amplified exponentially in vitro using two enzymes: reverse transcriptase (RT) and a DNA-dependent RNA polymerase (RNAP). A system has been constructed in which amplification of two molecular species is cooperatively coupled. These species are single-stranded (ss)DNA templates (D1 and D2) of lengths 58 and 68 nucleotides, respectively. Coupling occurs when D1 and D2 anneal to each other via a complementary region (DB and DB') situated at the 3' end of each template. RT elongates the hybridized templates producing a double-stranded (ds)DNA of 106 base pairs (bp). This double strand contains two promoters, which are situated on either side of, and directly adjacent to DB, and which are oriented towards each other. These promoters specify two RNA transcripts encompassing, respectively, the D1 and D2 portion of the dsDNA. After hybridization of two primers (P1 and P2) to the transcripts (R1 and R2) and reverse transcription, the ss templates D1 and D2 are regenerated. Amplification cycles of D1 and D2 are coupled cooperatively via the common dsDNA intermediate. Under optimized batch conditions the system shows the expected growth phases: exponential, linear and saturation phase. The enzymes of the 3SR cycle tend to misincorporate nucleotides and to produce abortive products. In future experiments, we intend to use the system for studies of evolutionary processes in spatially distributed systems where new strategies for optimization at the molecular level are possible.

Monitoring the amplification of CATCH, a 3SR based cooperatively coupled isothermal amplification system, by fluorimetric methods.

Three different types of fluorescence detection methods were employed to monitor amplification of a previously established isothermal cooperatively coupled amplification system as it can serve as a tool for the investigation of fundamental issues in evolutionary optimization. By using 5'IRD-41 fluorescent labeled primers, the intercalating dye TOPRO-1 and a 5'fluorescin/3'DABCYL 4-(4-dimethylamino-phenylazo)benzoic acid labeled ss 24 nt DNA, evolving molecular cooperation is accessible, sequence specifically as well as non-sequence-specifically without using radioactivity.

Structural dissection and functional analysis of the complex promoter of the streptokinase gene from Streptococcus equisimilis H46A.

The overlapping tandem promoters of the streptokinase gene, P1 and P2, identified previously by S1 nuclease transcript mapping were functionally dissected by mutagenesis of their -10 regions and fused transcriptionally with or without the 202-bp upstream region (USR) to the luciferase reporter gene (luc) from Photinus pyralis to analyze the contribution of the different sequence elements to promoter activity in Escherichia coli and the homologous Streptococcus equisimilis strain H46A. In E. coli, virtually the entire promoter activity derived from the upstream promoter P1. In S. equisimilis, luc expression increased in the following order of the involved sequence elements: P2 approximately equal to P2 + USR < P1 < P1 + P2 < P1 + USR < P1 + P2 + USR. This shows that (1) in the homologous system, P1 and P2 alone are extremely weak, (2) in the USR-less arrangement, only the combined core promoters have substantial activity, and (3) the USR stimulates only P1 and the combination of P1 + P2. Thus, the tandem promoters presumably function by mutual contributary action and their full activity strongly depends on the AT-rich and statically bent upstream region. The distinctive feature determining the strength of P1 in both hosts appears to be its extended -10 region which matches the consensus TRTGN established for strong S. pneumoniae and Bacillus subtilis promoters.

Complex transcriptional control of the streptokinase gene of Streptococcus equisimilis H46A.

On the Streptococcus equisimilis H46A chromosome, the divergent coding sequences of the genes for the plasminogen activator streptokinase (skc) and a leucine-rich protein (lrp), the function of which is unknown, are separated by a 328 bp intrinsically bent DNA region rich in AT tracts. To begin to understand the expression control of these two genes, we mapped their transcriptional initiation sites by S1 nuclease analysis and studied the influence of the bent intergenic region on promoter strength, using promoter-reporter gene fusions of skc' and lrp' to 'lacZ from Escherichia coli. The major transcriptional start sites, in both S. equisimilis and E. coli, mapped 22 bases upstream of the ATG start site of lrp (G), and 24 and 32 bases upstream of the translational initiation codon of skc (A and G, respectively), indicating the existence of two overlapping canonical skc promoters arranged in tandem on opposite faces of the helix. The reporter gene fusions were cloned in E. coli on a vector containing a 1.1 kb fragment of the S. equisimilis dexB gene, thus allowing promoter strength to be measured in multiple plasmid-form copies in the heterologous host and in single-copy genomic form following integration into the skc region of the homologous host. In S. equisimilis, skc'-'lacZ was expressed about 200-fold more strongly than the corresponding lrp'-'lacZ fusion. In contrast, in E. coli, the corresponding levels of expression differed by only about 11-fold. Deletion of the 202 bp bent region upstream of the skc and lrp core promoters caused a 13-fold decrease in skc promoter activity in S. equisimilis but did not alter lrp promoter strength in this host. In contrast, when studied in E. coli, this deletion did not alter the strength of the skc-double promoter and even increased by 2.4- to 3-fold the activity of the lrp promoter. This comparative promoter analysis shows that skc has a complex promoter structure, the activity of which in the homologous genomic environment specifically depends on sequences upstream of the two core promoters. Thus, the skc promoter structure resembles that of an array of promoters involved in a transcriptional switch; however, the nature of the potential switch factor(s) remains unknown.

The streptokinase gene: allelic variation, genomic environment and expression control.

The genes for streptokinase, the most important prokaryotic plasmingoen activator, exhibit allelic variation predominantly due to the polymorphism of an internal 220-base pair fragment that divides the phylogenetic tree of their products into two primary branches. Current molecular genetic research seeks functional correlates of the allelic variation, aims at analyzing the genomic environment of the streptokinase gene, skc, and focuses on understanding its expression. Of the six genes cloned and sequenced in the skc region of Streptococcus equisimilis H46A, skc is expressed most abundantly in a fashion that involves two overlapping core promoters and upstream sequences rich of AT tracts. Transcription of skc is terminated at a hypersymmetrical site that functions bidirectionally and prevents convergent transcription of the oppositely oriented skc and rel-orf1 genes whose mRNA abundance differs by a factor of at least three orders of magnitude.

Stalling of Escherichia coli RNA polymerase in the +6 to +12 region in vivo is associated with tight binding to consensus promoter elements.

Three synthetic promoters, PS1, PS2 and PS3, which differ in their core promoter elements, were studied in vivo and in vitro. Whereas an increased homology score correlates with higher rates of RNA polymerase binding, it does not correlate with activity in vivo. Permanganate probing in vivo reveals that PS1, which exhibits the lowest homology score, is rate-limited during the early phase of promoter-RNA polymerase interactions. By contrast, PS2 and PS3, with higher homology scores, are limited at a late step involving an open DNA region spanning from +6 to +12, indicating a stalling of RNA polymerase. These complexes disappear upon treatment of cells with rifampicin and are replaced by open complexes covering the start site. Because initiated complexes are selectively insensitive to rifampicin action, this confirms that RNA polymerase stalled at +6 to +12 has initiated RNA synthesis. Kinetic studies indicate that the enzyme is released slowly from this position and that this slow release appears to be responsible for the low promoter activity. For PS3, which exhibits the highest homology score and which binds RNA polymerase most efficiently, the release of the stalled complex is particularly slow. PS3 is found to be the weakest of the three promoters in vivo. These results support models in which promoter activity can be determined by various rate limiting steps, including those following the formation of open complexes and even the initiation of RNA synthesis.

Context-dependent effects of upstream A-tracts. Stimulation or inhibition of Escherichia coli promoter function.

Phased A-tract sequences were inserted in the upstream region of three synthetic promoters known to encompass different rate-limiting steps within the pathway of RNA polymerase-promoter interaction (Ellinger et al., accompanying paper). Promoter PS1, which is rate-limited in complex formation, was stimulated by A-tracts in vivo. Permanganate probing showed that the stimulation is due to an enhanced ability to compete for limiting RNA polymerase in vivo, leading to the increased formation of open complexes. By contrast, promoters PS2 and PS3, which are rate-limited in steps following open complex formation, were inhibited in vivo by A-tracts. Permanganate probing showed that the inhibition was accompanied by an A-tract-dependent accumulation of stalled initial transcribing complexes. A single A-tract was as effective as three. The phasing of the A-tracts with respect to the core promoter sequence was found to be important for promoter function. The position that caused maximal activation at one promoter caused maximal inhibition at another. These results suggest that the same molecular interaction gives rise to both inhibition and activation. This is likely to be due to facilitated RNA polymerase binding in the presence of A-tracts, which stimulates binding-limited promoters but inhibits promoter function in which polymerase escape and promoter clearance is rate limiting.