DNA looping and unlooping by AraC protein.

Expression of the L-arabinose BAD operon in Escherichia coli is regulated by AraC protein which acts both positively in the presence of arabinose to induce transcription and negatively in the absence of arabinose to repress transcription. The repression of the araBAD promoter is mediated by DNA looping between AraC protein bound at two sites near the promoter separated by 210 base pairs, araI and araO2. In vivo and in vitro experiments presented here show that an AraC dimer, with binding to half of araI and to araO2, maintains the repressed state of the operon. The addition of arabinose, which induces the operon, breaks the loop, and shifts the interactions from the distal araO2 site to the previously unoccupied half of the araI site. The conversion between the two states does not require additional binding of AraC protein and appears to be driven largely by properties of the protein rather than being specified by the slightly different DNA sequences of the binding sites. Slight reorientation of the subunits of AraC could specify looping or unlooping by the protein. Such a mechanism could account for regulation of DNA looping in other systems.

Transcription from the rha operon psr promoter.

S1 nuclease mapping experiments performed with RNA extracted from cell lines that were unable to metabolize L-rhamnose demonstrated that L-rhamnose and not a metabolite was the inducer of the L-rhamnose operons of Escherichia coli. In vitro transcription studies showed that purified RhaR activates transcription from the psr promoter in the presence of L-rhamnose. In the absence of L-rhamnose, RhaR binds to the psr promoter but does not activate transcription until L-rhamnose is added.

Equilibrium DNA-binding of AraC protein. Compensation for displaced ions.

Experiments on the AraC regulatory protein of Escherichia coli suggest a mechanism that DNA-binding proteins can use to reduce potentially drastic alterations in their affinity for DNA resulting from changes in salt concentration. Measurement of the net number of ions apparently displaced as AraC protein binds DNA and of fluorescence changes in the protein lead to the following picture. About 14 ions are displaced from the DNA as the protein binds the araI site. As the protein binds the DNA, however, it undergoes a conformational change and binds about ten ions. Consequently, the net order of the reaction is reduced from 15th to about fourth order in salt concentration.

Regulation of the Escherichia coli L-arabinose operon studied by gel electrophoresis DNA binding assay.

DNA binding properties of the proteins required for induction of the Escherichia coli L-arabinose operon were measured using a polyacrylamide gel electrophoresis assay. The mechanisms of induction and repression were studied by observing the multiple interactions of RNA polymerase, cyclic AMP receptor protein and araC protein with short DNA fragments containing either the araC or araBAD promoter regions. These studies show that binding of araC protein to the operator site, araO1, directly blocks RNA polymerase binding at the araC promoter, pC. We find that cyclic AMP receptor protein and araC protein do not bind co-operatively at their respective sites to linear DNA fragments containing the pBAD promoter. Nevertheless, both these positive effectors must be present on the DNA to stimulate binding of RNA polymerase. Additionally, binding of the proteins to the DNA is not sufficient; araC protein must also be in the inducing state, for RNA polymerase to bind. Equilibrium binding constraints and kinetics were determined for araC protein binding to the araI and the araO1 sites. In the presence of inducer, L-arabinose, araC protein binds with equal affinity to DNA fragments containing either of these sites. In the presence of anti-inducer, D-fucose, the affinity for both sites is reduced 40-fold. The apparent equilibrium binding constants for both states of the protein vary in parallel with the buffer salt concentration. This result suggests that the inducing and repressing forms of araC protein displace a similar number of cations upon binding DNA.

Purification and properties of RhaR, the positive regulator of the L-rhamnose operons of Escherichia coli.

The product of the rhaR gene, which regulates the level of mRNA produced from the four L-rhamnose-inducible promoters of the rhamnose operon, has been hypersynthesized and purified by a two-column procedure. The purified protein is a 33 kDa DNA-binding protein that binds to an inverted repeat structure located within the psr promoter, the promoter for the rhaS and rhaR genes. The equilibrium binding constants and kinetic constants have been determined under a variety of solution conditions. The protein binds with high affinity and its binding is sensitive to salt concentration and the presence of L-rhamnose. The nucleotides and phosphate residues contacted by RhaR were identified by chemical interference assays. All of the contacts are made to one face of the DNA and the symmetrical pattern matches the inverted repeat sequence proposed for the binding site. An unusual property of the binding site is that the two half-sites of the inverted repeat are separated from one another by 17 base-pairs of uncontacted DNA. Significant binding is retained if the 17 base-pairs are extended by insertions of integral turns of DNA, but not by half-integral turns. The complex of RhaR-DNA appears to be sharply bent, approximately 160 degrees.

AraC-DNA looping: orientation and distance-dependent loop breaking by the cyclic AMP receptor protein.

The arabinose operon promoter, pBAD, is negatively regulated in the absence of arabinose by AraC protein, which forms a DNA loop by binding to two sites separated by 210 base-pairs, araO2 and araI1. pBAD is also positively regulated by AraC-arabinose and the cyclic AMP receptor protein, CRP. We provide evidence that CRP breaks the araO2-araI1 repression loop in vitro. The ability of CRP to break the loop in vitro and to activate pBAD in vivo is dependent upon the orientation and distance of the CRP binding site relative to araI1. An insertion of one DNA helical turn, 11 base-pairs, between CRP and araI only partially inhibits CRP loop breaking and activation of pBAD, while an insertion of less than one DNA helical turn, 4 base-pairs, not only abolishes CRP activation and loop breaking, but actually causes CRP to stabilize the loop and increases the araO2-mediated repression of pBAD. Both integral and non-integral insertions of greater than one helical turn completely abolish CRP activation and loop breaking in vitro.

DNA-dependent renaturation of an insoluble DNA binding protein. Identification of the RhaS binding site at rhaBAD.

Previous work has indicated that the RhaS protein directly activates the L-rhamnose catabolic operon, rhaBAD, and that the likely RhaS binding site lies downstream of position -84 relative to the rhaBAD transcription start point. Biochemical analysis of RhaS binding to this DNA site had not been possible due to the extreme insolubility of overproduced RhaS protein. Here we have been able to analyze directly the DNA binding properties of RhaS by developing a method to refold insoluble RhaS protein into a form with specific DNA binding activity. We found that active RhaS protein could be recovered only if the renaturation reaction was performed in the presence of DNA. We also found that the recovery of DNA-binding activity from the related AraC protein, after denaturation in urea, was dependent upon added DNA. To test the specificity of the recovered RhaS DNA-binding activity, and to define the binding site for comparison with other AraC family binding sites, we then investigated the details of the RhaS binding site. Using refolded RhaS protein in a DNase footprinting assay, we found that RhaS protects a region of the rhaBAD promoter from position -83 to -28. Analysis of the effects of single base mutations in the rhaBAD promoter region indicates that RhaS binds to an inverted repeat of two 17 bp half-sites separated by 16 bp, located between -81 and -32 relative to the rhaBAD transcription start site.

Apo-AraC actively seeks to loop.

In the absence of arabinose and interactions with other proteins, AraC, the activator-repressor that regulates the araBAD operon in Escherichia coli, was found to prefer participating in DNA looping interactions between the two well-separated DNA half-sites, araI1 and araO2 at their normal separation of 211 base-pairs rather than binding to these same two half-sites when they are adjacent to one another. On the addition of arabinose, AraC preferred to bind to the adjacently located half-sites. Inverting the distally located araO2 half-site eliminated the looping preference. These results demonstrate that apo-AraC possesses an intrinsic looping preference that is eliminated by the presence of arabinose. We developed a method for the accurate determination of the relative affinities of AraC for the DNA half-sites araI1, araI2, and araO2 and non-specific DNA. These affinities allowed accurate calculation of basal level and induced levels of expression from pBAD under a wide variety of natural and mutant conditions. The calculations independently predicted the looping preference of apo-AraC.

Isolation and physical characterization of random insertions in Staphylococcal nuclease.

Using genetic engineering techniques we generated randomly located internal tandem duplications of random size within Staphylococcal nuclease. Those insertions, possessing greater than 0.1% of normal activity, were sequenced and characterized physically. Insertions were found to begin and end in regions possessing secondary structure as well as in regions without secondary structure. All proteins remained folded and monomeric, although one mutant appeared, by both circular dichroism and size exclusion chromatography, to be partially unfolded. The stability of the insertions as assayed by guanidine hydrochloride denaturation ranged from nearly normal to destabilized by almost 4 kcal per mol. The activities of the insertion mutants ranged from 1/30 to 1/2000 of the parental nuclease.

Transcription start site and induction kinetics of the araC regulatory gene in Escherichia coli K-12.

The in vivo transcription start site of the araC message was determined by S1 nuclease mapping of hybrids formed between labeled DNA, and RNA extracted from cells grown under a variety of physiological conditions, including the interval of transient derepression following arabinose addition. Under all conditions tested, transcription initiated from the same nucleotide position at -148.

A regulatory cascade in the induction of rhaBAD.

The RhaS and RhaR regulatory proteins are encoded in the Escherichia coli L-rhamnose gene cluster. We used complementation analysis and DNA mobility shift assays to show that RhaR is not the direct activator of the L-rhamnose catabolic operon, rhaBAD. An in-frame deletion of rhaS (rhaS-rhaR+) eliminated expression from the rhaBAD promoter, pBAD, while overexpression of rhaS greatly speeded the normally slow induction of transcription from pBAD. Expression from pBAD in a coupled transcription-translation assay was only detected when rhaS+ DNA was added to allow synthesis of RhaS protein. RhaS thus appears to be the direct L-rhamnose-specific activator of rhaBAD expression. Deletion mapping located the binding site for the L-rhamnose-specific regulator to a region overlapping position -70 relative to the rhaBAD transcription start site. Deletion mapping and DNA mobility shift assays located a CRP binding site just upstream from the binding site for the L-rhamnose-specific regulator. Quantitative primer extension analysis showed that induction of both the rhaBAD and rhaSR messages was unusually slow, requiring 40 to 50 minutes to reach a steady-state level. Induction of rhaBAD apparently involves a regulatory cascade in which RhaR first induces rhaSR expression, then RhaS accumulates and induces rhaBAD expression.

Positive regulation of the Escherichia coli L-rhamnose operon is mediated by the products of tandemly repeated regulatory genes.

The rhaC gene, whose product is the positive activator of the genes required for L-rhamnose utilization, has been cloned along with the rhamnose structural genes. The rhaC sequence shows two partially overlapping reading frames, encoding two proteins of molecular weight 32,000 and 35,000 RhaS and RhaR. Both proteins show significant homology to AraC, the positive activator of the arabinose operon. S1 mapping located transcriptional start points and showed that RhaR, and possibly RhaS, positively regulate transcription from the structural gene promoters as well as transcription from their own promoter. In-vivo dimethyl sulfate footprinting and DNase I footprinting indicate that the RhaR protein may bind to DNA elements upstream from its RNA polymerase binding site.

Hemiplegic mutations in AraC protein.

We have isolated mutations in AraC protein that specifically block either induction or repression at the ara pBAD promoter. These hemiplegic mutations identify amino acid residues that, correspondingly, are involved only in the induction or only in the repression activities of the protein. Residues key only for induction are 13, 15, and 18, which are located in the N-terminal arm of AraC, and residues 80 and 82 which lie in the arabinose-binding pocket of the protein's sugar-binding and dimerization domain. Alteration of residues 157, 244 and 257 can leave the protein able to activate transcription but not able to repress transcription. The behavior of the mutant proteins is consistent with the light switch mechanism for AraC action in which the presence of arabinose pulls the N-terminal arms of the protein off the DNA-binding domains, thereby freeing them to assume a direct-repeat orientation, bind to adjacent direct-repeat DNA half-sites, and activate transcription.

Arm-domain interactions in AraC.

N-terminal deletions extending beyond the sixth amino acid of the Escherichia coli regulator of the l-arabinose operon, AraC, were found to generate constitutive regulatory behavior of the promoter pBAD. Mutagenesis of the DNA coding for the first 20 amino acids of the protein and screening for constitutives yielded mutants across the region whereas screening for mutants that cannot induce pBAD, even in the presence of arabinose, yielded none. These results indicate that the N-terminal arm is not essential for transcription activation, but that it plays an important and active role in holding the system in a non-activating state. Despite the fact that arabinose binds to the N-terminal domain of AraC, mutations were found in the C-terminal domain that weaken the binding of arabinose to the protein. The effects of the mutations could be suppressed by specific mutation in the N-terminal arm or by deletion of the arm. These results, in conjunction with the crystal structures of the N-terminal domain determined in the presence and absence of arabinose, indicate that in the absence of arabinose, the N-terminal arms of the protein bind to the C-terminal DNA binding domains to hold them in a state where the protein prefers to loop. When arabinose is added, the arms are pulled off the C-terminal domains, thereby releasing them to bind to adjacently located DNA half-sites and activate transcription.

Reaching out. Locating and lengthening the interdomain linker in AraC protein.

A genetic method was developed to determine, in proteins, areas which are tolerant of insertions and deletions. Attractive candidates for these areas are linker regions. Such a region was found to include positions 171 to 178 in the Escherichia coli regulatory protein AraC. Independent biochemical methods identified amino acid residues 11 to 170 as the minimal dimerization domain of AraC, and amino acid residues 178 to 286 out of the 291 residue protein as the minimal DNA-binding domain. Hence, by both the genetic and biochemical approaches, the interdomain linking region was determined to include amino acid residues 171 to 177. The properties of altered proteins were examined using templates with AraC half-sites more widely separated than in the wild-type case. Both AraC protein containing an insertion in the interdomain linker region and a protein consisting of the minimal functional dimerization and DNA-binding domains separated by a 39 amino acid residue linker were able to bind to and function on such a DNA site. In vitro, the proteins with longer linkers bound substantially more stably than wild-type AraC to the DNA containing half-sites for AraC separated by an extra two helical turns of DNA. In vivo on an ara promoter with the more widely separated AraC half-sites, the proteins could activate transcription much better than wild-type AraC.

In vivo DNA loops in araCBAD: size limits and helical repeat.

Formation of a DNA loop by AraC proteins bound at the araI and araO2 sites, whose center-to-center distance is 211 base pairs, is necessary for repression of the araBAD promoter, PBAD, of Escherichia coli. To determine the upper and lower size limits of the loop, we constructed PBAD-reporter gene fusion plasmids with various spacings between araI and araO2 and measured their levels of expression. Spacings larger than about 500 base pairs resulted in elimination of detectable repression. No lower limit to spacing was found, suggesting that AraC protein itself possesses significant flexibility and its bending substantially aids formation of small loops. As the spacing between araI and araO2 varied, the activity of PBAD oscillated with an 11.1-base-pair periodicity, implying that the in vivo helical repeat of this DNA is 11.1 base pairs per turn.

The linker region of AraC protein.

AraC protein, a transcriptional regulator of the L-arabinose operon in Escherichia coli, is dimeric. Each monomer consists of a domain for DNA binding plus transcription activation and a domain for dimerization plus arabinose binding. These are connected to one another by a linker region of at least 5 amino acids. Here we have addressed the question of whether any of the amino acids in the linker region play active, specific, and crucial structural roles or whether these amino acids merely serve as passive spacers between the functional domains. We found that all but one of the linker amino acids can be changed to other amino acids individually and in small groups without substantially affecting the ability of AraC protein to activate transcription when arabinose is present. When, however, the entire linker region is replaced with linker sequences from other proteins, the functioning of AraC is impaired.

In vivo association of protein fragments giving active AraC.

Frameshift mutations in a restricted portion of the arabinose operon regulatory gene araC from Escherichia coli give rise to active AraC protein, likely from the in vivo synthesis of two incomplete fragments that are active together. Synthesis of corresponding fragments, each separately inactive, from two plasmids within cells also resulted in complementation.