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.

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.

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.

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.

Interaction at a distance between lambda repressors disrupts gene activation.

The lambda repressor is an activator as well as a repressor of transcription. The activation function is blocked by interaction with another lambda repressor molecule bound upstream on the same DNA molecule. This example of negative control at a distance involves formation of a DNA loop.

Amino acid substitutions in the -35 recognition motif of sigma 70 that result in defects in phage lambda repressor-stimulated transcription.

The phage lambda repressor activates transcription of its own gene from the promoter PRM. Previous work has suggested that this activation involves a protein-protein interaction between DNA-bound repressor and RNA polymerase. To identify the subunit of RNA polymerase that participates in this putative interaction, we searched for polymerase mutants that responded poorly to repressor. We report here the isolation of three sigma mutants that caused defects in repressor-stimulated, but not basal, transcription from PRM. These mutants bear amino acid substitutions in a putative helix-turn-helix motif that sigma uses to recognize the promoter -35 region. We suggest that lambda repressor interacts directly with this helix-turn-helix motif in facilitating the formation of a productive initiating complex.

Homologous interactions of lambda repressor and lambda Cro with the lambda operator.

Lambda repressor and lambda Cro bind to the same six sites on the phage chromosome but with different relative affinities. Nucleotides at certain positions in the operator are conserved in all sites, as are amino acids at certain positions in the recognition alpha-helices of repressor and Cro. Here we focus on one of the conserved amino acids, a serine found at position 2 of each recognition helix. We show that, contrary to a previous model, both serines contact the same conserved position in the operator, position 4. We suggest a simplified view of how repressor and Cro recognized similar operator sites but distinguish differently among them.

Cooperative binding of lambda repressors to sites separated by integral turns of the DNA helix.

Lambda repressors bind cooperatively to adjacent pairs of operator sites. Here we show that repressors bind cooperatively to pairs of operator sites whose centers have been separated by five or six turns of the helix. No cooperativity is observed when the centers of these sites are on opposite sides of the DNA helix. Cooperativity depends upon the same part of the protein (the carboxyl domain) that mediates cooperativity when the sites are adjacent. As the repressors bind, the DNA between the sites becomes alternately sensitive and resistant to DNAase I cleavage at half turn intervals. We suggest that when repressors bind cooperatively to separated sites, the DNA forms a loop, thus allowing the two repressors to touch.

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 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.

How lambda repressor and lambda Cro distinguish between OR1 and OR3.

Although lambda repressor and lambda Cro bind to the same six operators on the phage chromosome, the fine specificities of the two proteins differ: repressor binds more tightly to OR1 than to OR3, and vice versa for Cro. In this paper, we change base pairs in the operators and amino acids in the proteins to analyze the basis for these preferences. We find that these preferences are determined by residues 5 and 6 of the recognition helices of the two proteins and by the amino-terminal arm, in the case of repressor. We also find that the most important base pairs in the operator which enable repressor and Cro to discriminate between OR1 and OR3 are position 3 (for Cro) and positions 5 and 8 (for repressor). These and previous results show how repressor and Cro recognize and distinguish between two related operator sequences.

Repressor structure and the mechanism of positive control.

It has been suggested that the lambda repressor stimulates transcription of its own gene by binding to the lambda operator and contacting RNA polymerase bound to the adjacent promoter. We describe three different mutants (called pc) of the lambda phage repressor that are specifically deficient in the positive control function. We show that the amino acid residues altered in the pc mutants lie on the surface of the DNA-bound repressor that we predict, based on structural and other evidence, would most closely approach DNA-bound polymerase. Furthermore, we describe a pc mutant of the P22 repressor. We argue that in both the lambda and P22 repressors a structure comprised of two alpha helices has two functions: to bind DNA and to contact RNA polymerase. In the two cases, however, different regions of this structure contact polymerase to mediate positive control.

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.

DNA loops induced by cooperative binding of lambda repressor.

It has been shown by Hochschild and Ptashne that lambda repressors bind cooperatively to operator sites separated by five or six turns of the helix. Cooperative binding is not observed if the sites are separated by a nonintegral number of turns, unless a four-nucleotide gap is introduced into one of the strands between the two sites. These and other facts suggested that repressors at the separated sites touch each other, the DNA bending smoothly so as to accommodate the protein-protein interaction. Here we use electron microscopy to visualize the predicted protein-DNA complexes.

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.

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.

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.

Synergistic activation of transcription by Escherichia coli cAMP receptor protein.

Activation of gene expression in eukaryotes generally involves the action of multiple transcription factors that function synergistically when bound near a particular target gene. Such effects have been suggested to occur because multiple activators can interact simultaneously with one or more components of the basal transcription machinery. In prokaryotes, examples of synergistic effects on transcription are much more limited and can often be explained by cooperative DNA binding. Here we show that the Escherichia coli cAMP receptor protein (CRP) functions synergistically to activate transcription from a derivative of the lac promoter that bears a second CRP-binding site upstream of the natural binding site. We present evidence indicating that cooperative DNA binding of two CRP dimers does not account for the magnitude of the observed cooperative activation. We suggest, instead, that the two dimers stimulate transcription directly by contacting two distinct surfaces of RNA polymerase simultaneously. Thus, synergistic activation by CRP may provide a relatively simple model for examining the molecular basis of such effects in higher organisms.