A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme.

The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the beta subunit and conserved region 4 of the sigma subunit, is a potential target for regulation. Here we provide genetic and biochemical evidence that the sigma region 4/beta-flap interaction is targeted by the transcription factor AsiA. Specifically, we show that AsiA competes directly with the beta-flap for binding to sigma region 4, thereby inhibiting transcription initiation by disrupting the sigma region 4/beta-flap interaction.

Bacterial two-hybrid analysis of interactions between region 4 of the sigma(70) subunit of RNA polymerase and the transcriptional regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa.

A number of transcriptional regulators mediate their effects through direct contact with the sigma(70) subunit of Escherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of sigma(70) that harbors conserved region 4. This region of sigma contains a putative helix-turn-helix DNA-binding motif that contacts the -35 element of sigma(70)-dependent promoters directly. Here we report the use of a recently developed bacterial two-hybrid system to study the interaction between the putative anti-sigma factor Rsd and the sigma(70) subunit of E. coli RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of sigma(70) and also that amino acid substitution R596H, within region 4 of sigma(70), weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between sigma and two other regulators shown previously to contact region 4 of sigma(70). We also demonstrated that AlgQ, a homolog of Rsd that positively regulates virulence gene expression in Pseudomonas aeruginosa, can contact the C-terminal region of the sigma(70) subunit of RNAP from this organism. We found that amino acid substitution R600H in sigma(70) from P. aeruginosa, corresponding to the R596H substitution in E. coli sigma(70), specifically weakens the interaction between AlgQ and sigma(70). Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of sigma(70) and probably regulate gene expression through this contact.

A chimeric activator of transcription that uses two DNA-binding domains to make simultaneous contact with pairs of recognition sites.

Many well-known transcriptional regulatory proteins are composed of at least two independently folding domains and, typically, only one of these is a DNA-binding domain. However, some transcriptional regulators have been described that have more than one DNA-binding domain. Regulators with a single DNA-binding domain often bind co-operatively to the DNA in homotypic or heterotypic combinations, and two or more DNA-binding domains of a single regulatory protein can also bind co-operatively to suitably positioned recognition sequences. Here, we examine the behaviour of a chimeric activator of transcription with two different DNA-binding domains, that of the bacteriophage lambda cI protein and that of the Escherichia coli cyclic AMP receptor protein. We show that these two DNA-binding moieties, when present in the same molecule, can bind co-operatively to a pair of cognate recognition sites located upstream of a test promoter, thereby permitting the chimera to function as a particularly strong activator of transcription from this promoter. Our results show how such a bivalent DNA-binding protein can be used to regulate transcription differentially from promoters that bear either one or both recognition sites.

Magnitude of the CREB-dependent transcriptional response is determined by the strength of the interaction between the kinase-inducible domain of CREB and the KIX domain of CREB-binding protein.

The activity of the transcription factor CREB is regulated by extracellular stimuli that result in its phosphorylation at a critical serine residue, Ser133. Phosphorylation of Ser133 is believed to promote CREB-dependent transcription by allowing CREB to interact with the transcriptional coactivator CREB-binding protein (CBP). Previous studies have established that the domain encompassing Ser133 on CREB, known as the kinase-inducible domain (KID), interacts specifically with a short domain in CBP termed the KIX domain and that this interaction depends on the phosphorylation of Ser133. In this study, we adapted a recently described Escherichia coli-based two-hybrid system for the examination of phosphorylation-dependent protein-protein interactions, and we used this system to study the kinase-induced interaction between the KID and the KIX domain. We identified residues of the KID and the KIX domain that are critical for their interaction as well as two pairs of oppositely charged residues that apparently interact at the KID-KIX interface. We then isolated a mutant form of the KIX domain that interacts more tightly with wild-type and mutant forms of the KID than does the wild-type KIX domain. We show that in the context of full-length CBP, the corresponding amino acid substitution resulted in an enhanced ability of CBP to stimulate CREB-dependent transcription in mammalian cells. Conversely, an amino acid substitution in the KIX domain that weakens its interaction with the KID resulted in a decreased ability of full-length CBP to stimulate CREB-dependent transcription. These findings demonstrate that the magnitude of CREB-dependent transcription in mammalian cells depends on the strength of the KID-KIX interaction and suggest that the level of transcription induced by coactivator-dependent transcriptional activators can be specified by the strength of the activator-coactivator interaction.

Mechanism for a transcriptional activator that works at the isomerization step.

Transcriptional activators in prokaryotes have been shown to stimulate different steps in the initiation process including the initial binding of RNA polymerase (RNAP) to the promoter and a postbinding step known as the isomerization step. Evidence suggests that activators that affect initial binding can work by a cooperative binding mechanism by making energetically favorable contacts with RNAP, but the mechanism by which activators affect the isomerization step is unclear. A well-studied example of an activator that normally exerts its effect exclusively on the isomerization step is the bacteriophage lambda cI protein (lambdacI), which has been shown genetically to interact with the C-terminal region of the final sigma(70) subunit of RNAP. We show here that the interaction between lambdacI and final sigma can stimulate transcription even when the relevant portion of final sigma is transplanted to another subunit of RNAP. This activation depends on the ability of lambdacI to stabilize the binding of the transplanted final sigma moiety to an ectopic -35 element. Based on these and previous findings, we discuss a simple model that explains how an activator's ability to stabilize the binding of an RNAP subdomain to the DNA can account for its effect on either the initial binding of RNAP to a promoter or the isomerization step.

Crystal structure of the lambda repressor C-terminal domain provides a model for cooperative operator binding.

Interactions between transcription factors bound to separate operator sites commonly play an important role in gene regulation by mediating cooperative binding to the DNA. However, few detailed structural models for understanding the molecular basis of such cooperativity are available. The c1 repressor of bacteriophage lambda is a classic example of a protein that binds to its operator sites cooperatively. The C-terminal domain of the repressor mediates dimerization as well as a dimer-dimer interaction that results in the cooperative binding of two repressor dimers to adjacent operator sites. Here, we present the x-ray crystal structure of the lambda repressor C-terminal domain determined by multiwavelength anomalous diffraction. Remarkably, the interactions that mediate cooperativity are captured in the crystal, where two dimers associate about a 2-fold axis of symmetry. Based on the structure and previous genetic and biochemical data, we present a model for the cooperative binding of two lambda repressor dimers at adjacent operator sites.

Isolation of peptide aptamers that inhibit intracellular processes.

We have developed a method for isolation of random peptides that inhibit intracellular processes in bacteria. A library of random peptides expressed as fusions to Escherichia coli thioredoxin (aptamers) were expressed under the tight control of the arabinose-inducible P(BAD) promoter. A selection was applied to the library to isolate aptamers that interfered with the activity of thymidylate synthase (ThyA) in vivo. Expression of an aptamer isolated by this method resulted in a ThyA(-) phenotype that was suppressed by simultaneous overexpression of ThyA. Two-hybrid analysis showed that this aptamer is likely to interact with ThyA in vivo. The library also was screened for aptamers that inhibited growth of bacteria expressing them, and five such aptamers were characterized. Four aptamers were bacteriostatic when expressed, whereas one showed a bactericidal effect. Introduction of translational stop codons into various aptamers blocked their activity, suggesting that their biological effects were likely to be due to protein aptamer rather than RNA. Combinatorial aptamers provide a new genetic and biochemical tool for identifying targets for antibacterial drug development.

Escherichia coli one- and two-hybrid systems for the analysis and identification of protein-protein interactions.

Genetic methods based on fusion proteins allow the power of a genetic approach to be applied to the self-assembly of proteins or protein fragments, regardless of whether or not the normal function of the fused assembly domains is either known or amenable to selection or screening. The widespread adoption of variations of the yeast two-hybrid system originally described by S. Fields and O. Song (1989, Nature 340, 245-246) demonstrates the usefulness of these kinds of assays. This review describes some of the many systems used to select or screen for protein-protein interactions based on the regulation of reporter constructs by hybrid proteins expressed in bacteria, including recent implementations of generalizable two-hybrid systems for Escherichia coli.

A cyclic AMP receptor protein mutant that constitutively activates an Escherichia coli promoter disrupted by an IS5 insertion.

Previously an Escherichia coli mutant that had acquired the ability to grow on propanediol as the sole carbon and energy source was isolated. This phenotype is the result of the constitutive expression of the fucO gene (in the fucAO operon), which encodes one of the enzymes in the fucose metabolic pathway. The mutant was found to bear an IS5 insertion in the intergenic regulatory region between the divergently oriented fucAO and fucPIK operons. Though expression of the fucAO operon was constitutive, the fucPIK operon became noninducible such that the mutant could no longer grow on fucose. A fucose-positive revertant which was found to contain a suppressor mutation in the crp gene was selected. Here we identify this crp mutation, which results in a single amino acid substitution (K52N) that has been proposed previously to uncover a cryptic activating region in the cyclic AMP receptor protein (CRP). We show that the mutant CRP constitutively activates transcription from both the IS5-disrupted and the wild-type fucPIK promoters, and we identify the CRP-binding site that is required for this activity. Our results show that the fucPIK promoter, a complex promoter which ordinarily depends on both CRP and the fucose-specific regulator FucR for its activation, can be activated in the absence of FucR by a mutant CRP that uses three, rather than two, activating regions to contact RNA polymerase. For the IS5-disrupted promoter, which retains a single CRP-binding site, the additional activating region of the mutant CRP evidently compensates for the lack of upstream regulatory sequences.

A genetic method for dissecting the mechanism of transcriptional activator synergy by identical activators.

Pairs of transcriptional activators in prokaryotes have been shown to activate transcription synergistically from promoters with two activator binding sites. In some cases, such synergistic effects result from cooperative binding, but in other cases each DNA-bound activator plays a direct role in the activation process by interacting simultaneously with separate surfaces of RNA polymerase. In such cases, each DNA-bound activator must possess a functional activating region, the surface that mediates the interaction with RNA polymerase. When transcriptional activation depends on two or more identical activators, it is not straightforward to test the requirement of each activator for a functional activating region. Here we describe a method for directing a mutationally altered activator to either one or the other binding site, and we demonstrate the use of this method to examine the mechanism of transcriptional activator synergy by the Escherichia coli cyclic AMP receptor protein (CRP) working at an artificial promoter bearing two CRP-binding sites.

Amino acid-amino acid contacts at the cooperativity interface of the bacteriophage lambda and P22 repressors.

The bacteriophage lambda repressor and its relatives bind cooperatively to adjacent as well as artificially separated operator sites. This cooperativity is mediated by a protein-protein interaction between the DNA-bound dimers. Here we use a genetic approach to identify two pairs of amino acids that interact at the dimer-dimer interface. One of these pairs is nonconserved in the aligned sequences of the lambda and P22 repressors; we show that a lambda repressor variant bearing the P22 residues at these two positions interacts specifically with the P22 repressor. The other pair consists of a conserved ion pair; we reverse the charges at these two positions and demonstrate that, whereas the individual substitutions abolish the interaction of the DNA-bound dimers, these changes in combination restore the interaction of both lambdacI and P22c2 dimers.

Conversion of the omega subunit of Escherichia coli RNA polymerase into a transcriptional activator or an activation target.

Evidence obtained in both eukaryotes and prokaryotes indicates that arbitrary contacts between DNA-bound proteins and components of the transcriptional machinery can activate transcription. Here we demonstrate that the Escherichia coli omega protein, which copurifies with RNA polymerase, can function as a transcriptional activator when linked covalently to a DNA-binding protein. We show further that omega can function as an activation target when this covalent linkage is replaced by a pair of interacting polypeptides fused to the DNA-binding protein and to omega, respectively. Our findings imply that the omega protein is associated with RNA polymerase holoenzyme in vivo, and provide support for the hypothesis that contact between a DNA-bound protein and any component of E. coli RNA polymerase can activate transcription.

Activation of prokaryotic transcription through arbitrary protein-protein contacts.

Many transcriptional activators in prokaryotes are known to bind near a promoter and contact RNA polymerase, but it is not clear whether a protein-protein contact between an activator and RNA polymerase is enough to activate gene transcription. Here we show that contact between a DNA-bound protein and a heterologous protein domain fused to RNA polymerase can elicit transcriptional activation; moreover, the strength of this engineered protein-protein interaction determines the amount of gene activation. Our results indicate that an arbitrary interaction between a DNA-bound protein and RNA polymerase can activate transcription. We also find that when the DNA-bound 'activator' makes contact with two different components of the polymerase, the effect of these two interactions on transcription is synergistic.

The activation defect of a lambda cI positive control mutant.

"Positive control" mutants of the cI protein of bacteriophage lambda (lambda cI) bind DNA but, unlike the wild-type protein, fail to activate transcription. According to the original interpretation of Ptashne and co-workers, these mutants bear amino acid substitutions that disrupt a stimulatory interaction between lambda cI bound at operator site O(R)2 and RNA polymerase bound at promoter P(RM), an idea supported by kinetic analysis in one case. Genetic analysis has suggested that one residue in particular, glutamate 34 (E34), is critical for the stimulatory effect of wild-type lambda cI. More recently, however, Kolkhof and Muller-Hill have challenged this view, suggesting that mutant E34K fails to activate because it binds at unusually low concentrations to O(R)3, a site that mediates repression of P(RM). To test this hypothesis, we have examined the behaviour of the lambda cI-E34K mutant both in vitro and in vivo by assaying transcription from P(RM) and monitoring operator site occupancy over a range of protein concentrations. Our results are inconsistent with the interpretation of Kolkhof and Muller-Hill, and demonstrate that under conditions where lambda operator O(R)2 is fully occupied and operator O(R)3 is vacant, wild-type lambda cI activates transcription from promoter P(RM) whereas the mutant does not.

Genetic strategy for analyzing specificity of dimer formation: Escherichia coli cyclic AMP receptor protein mutant altered in its dimerization specificity.

Many transcriptional regulators function in homo- or heterodimeric combinations. The same protein can carry out distinct regulatory functions depending on the partner with which it associates. Here, we describe a mutant of the Escherichia coli cAMP receptor protein (CRP) that has an altered dimerization specificity; that is, mutant/mutant homodimers form preferentially over wild-type/mutant heterodimers. CRP dimerization involves the formation of a parallel coiled-coil structure, and our CRP mutant bears an amino acid substitution affecting the first "d" position residue within the alpha-helix that mediates CRP dimerization. The genetic strategy we used to isolate this CRP altered dimerization specificity (ADS) mutant is generalizable and could be utilized to isolate ADS mutants of other dimeric transcriptional regulators.

Synergistic activation of transcription by bacteriophage lambda cI protein and E. coli cAMP receptor protein.

Two heterologous prokaryotic activators, the bacteriophage lambda cI protein (lambda cI) and the Escherichia coli cyclic AMP receptor protein (CRP), were shown to activate transcription synergistically from an artificial promoter bearing binding sites for both proteins. The synergy depends on a functional activation (positive control) surface on each activator. These results imply that both proteins interact directly with RNA polymerase and thus suggest a precise mechanism for transcriptional synergy: the interaction of two activators with two distinct surfaces of RNA polymerase.

Specificity determinants for the interaction of lambda repressor and P22 repressor dimers.

The related phage lambda and phage P22 repressors each bind cooperatively to adjacent and separated operator sites, an interaction that involves a pair of repressor dimers. The specificities of these interactions differ: Each dimer interacts with its own type but not with dimers of the heterologous repressor. The two repressors exhibit significant amino acid sequence homology in their carboxy-terminal domains, which are responsible for both dimer formation and the dimer-dimer interaction. Here, we identify a collection of amino acid substitutions that disrupt the protein-protein interaction of DNA-bound lambda repressor dimers and show that several of these substitutions have the same effect when introduced at the corresponding positions of P22 repressor. We use this information to construct a variant of the lambda repressor bearing only six non-wild-type amino acids that has a switched specificity; that is, it binds cooperatively with P22 repressor, but not with wild-type lambda repressor. These results identify a series of residues that determine the specificities of the two interactions.

Transcriptional activation. How lambda repressor talks to RNA polymerase.

The bacteriophage lambda repressor protein can activate or repress transcription. Amino-acid substitutions in the sigma subunit of RNA polymerase affect repressor-stimulated transcription, shedding light on the activation process.