Biomedical subjects

R F Schleif

Publications and source records attributed to R F Schleif.

At least 19 recordsLinked to original sources

PubMed2001

Biophysical evidence of arm-domain interactions in AraC.

We report development of a method for the direct measurement of the interaction between the N-terminal arm and the remainder of the dimerization domain in the Escherichia coli AraC protein, the regulator of the l-arabinose operon. The interaction was measured using surface plasmon resonance to monitor the association between the immobilized peptide arm and the dimerization domain, truncated of its arm, in solution. As expected from genetic and physiological data, the interaction is strongly stimulated by l-arabinose and is insensitive to sugars like d-glucose or d-galactose. Alterations in the sequence of the arm which physiological experiments predict either to strengthen or weaken the arm produce the expected responses.

Amino Acid Sequence↗

PubMed2001

Stabilizing C-terminal tails on AraC.

We examined the effects of the metabolic stability of random sequences appended to the C-terminus of the dimerization domain of the regulatory protein of the Escherichia coli arabinose operon, AraC. Genetic scoring utilized the trans dominant negative effect of the dimerization domain on the activity of intact AraC, and physical scoring used sodium dodecyl sulfate (SDS) gel electrophoresis. We confirmed previous results obtained with Arc and lambda repressors that C-terminal charged residues tend to be stabilizing and that hydrophobic residues are destabilizing. Additionally, we found that the provision of a single, charged C-terminal residue conferred significant stability that was independent of interior sequence. Hence, it appears that in the engineering of proteins, flexible tails may be freely added, with only the identity of the C-terminal amino acid being restricted. Proteins 2001;42:177-181.

Amino Acid Sequence↗

PubMed2000

Cooperative action of the catabolite activator protein and AraC in vitro at the araFGH promoter.

Full activation of transcription of the araFGH promoter, p(FGH), requires both the catabolite activator protein (CAP) and AraC protein. At p(FGH), the binding site for CAP is centered at position -41.5, an essential binding site for AraC is centered at position -79.5, and a second, nonessential binding site is centered at position -154.5. In this work, we used the minimal promoter region required for in vivo activation of p(FGH) to examine the roles of CAP and AraC in stimulating formation of open complexes at p(FGH). Migration retardation assays of open complexes showed that RNA polymerase binds exceptionally tightly to the AraC-CAP-p(FGH) complex and that the order of addition of proteins to the initiating complex is important. Similar assays with RNA polymerase containing truncated alpha subunits suggest that AraC interacts with the C-terminal domain of the alpha subunit. Finally, AraC protein also acts to prevent the improper binding of RNA polymerase at a pseudo promoter near the true p(FGH) promoter.

Allosteric Regulation↗

PubMed1999

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.

AraC Transcription Factor↗

PubMed1998

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.

Chromatography, Gel↗

PubMed1998

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.

AraC Transcription Factor↗

PubMed1998

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.

Apoproteins↗

PubMed1998

DNA bending by AraC: a negative mutant.

We sought a mutation in the DNA binding domain of the arabinose operon regulatory protein, AraC, of Escherichia coli that allows the protein to bind DNA normally but not activate transcription. The mutation was isolated by mutagenizing a plasmid overproducing a chimeric leucine zipper-AraC DNA binding domain and screening for proteins that were trans dominant negative with regard to wild-type AraC protein. The mutant with the lowest transcription activation of the araBAD promoter was studied further. It proved to alter a residue that had previously been demonstrated to contact DNA. Because the overproduced mutant protein still bound DNA in vivo, it is deficient in transcription activation for some reason other than absence of DNA binding. Using the phase-sensitive DNA bending assay, we found that wild-type AraC bends DNA about 90 degrees whereas the mutant bends DNA by a smaller amount.

AraC Transcription Factor↗

PubMed1996

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.

AraC Transcription Factor↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1995

In vivo induction kinetics of the arabinose promoters in Escherichia coli.

In Escherichia coli, the AraC protein represses transcription from its own promoter, PC, and when associated with arabinose, activates transcription from three other promoters, PBAD, PE, and PFGH. Expression from all four of these promoters is also regulated by cyclic AMP-catabolite activator protein; however, the arrangement of the protein binding sites is not identical for each promoter. We are interested in determining how the AraC protein is able to activate PBAD, PE, and PFGH despite their differences. We have characterized the induction response of the wild-type arabinose operons from their native chromosomal locations by primer extension analysis. In this analysis, mRNA from the four arabinose operons plus an internal standard could all be assayed in the RNA obtained from a single sample of cells. We found that each of the operons shows a rapid, within 15 to 30 s, response to arabinose. We also found that the expression of araFGH is more sensitive to catabolite repression but not to arabinose concentration than are araE and araBAD. Finally, we have determined the relative levels of inducibility in wild-type cells of araBAD, araFGH, and araE to be 6.5, 5, and 1, respectively. These results provide a basis for subsequent studies to determine the mechanism(s) by which AraC protein activates transcription from the different arabinose promoters.

AraC Transcription Factor↗

PubMed1994

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.

AraC Transcription Factor↗

PubMed1994

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.

Amino Acid Sequence↗

PubMed1993

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.

Bacterial Proteins↗

PubMed1993

Functional domains of the AraC protein.

The AraC protein, which regulates the L-arabinose operons in Escherichia coli, was dissected into two domains that function in chimeric proteins. One provides a dimerization capability and binds the ligand arabinose, and the other provides a site-specific DNA-binding capability and activates transcription. In vivo and in vitro experiments showed that a fusion protein consisting of the N-terminal half of the AraC protein and the DNA-binding domain of the LexA repressor dimerizes, binds well to a LexA operator, and represses expression of a LexA operator-beta-galactosidase fusion gene in an arabinose-responsive manner. In vivo and in vitro experiments also showed that a fusion protein consisting of the C-terminal half of the AraC protein and the leucine zipper dimerization domain from the C/EBP transcriptional activator binds to araI and activates transcription from a PBAD promoter-beta-galactosidase fusion gene. Dimerization was necessary for occupancy and activation of the wild-type AraC binding site.

AraC Transcription Factor↗

PubMed1993

Formation of AraC-DNA sandwiches.

With the use of special DNA binding sites, but not the natural aral binding site, the dimeric AraC protein can be forced to make sandwich structures in which two DNA molecules are joined by two AraC protein dimers. Apparently one subunit from each dimer contacts each DNA molecule in an extended structure. These sandwich structures form only in the absence of arabinose. This behavior is consistent with the protein's ability to form DNA loops by binding to separated half sites in the absence of arabinose and its preference for binding to adjacent half-sites in the presence of arabinose.

AraC Transcription Factor↗

PubMed1993

Variation of half-site organization and DNA looping by AraC protein.

The dimeric AraC protein of Escherichia coli binds specifically to DNA sequences upstream of promoters whose transcription is regulated by arabinose. Here we show with affinity measurements, DNase footprinting, dimethyl sulfate premethylation interference and dimethyl sulfate footprinting studies that AraC protein can recognize paired half-sites in direct repeat orientation or inverted repeat orientation. A similar high degree of flexibility was also seen in the ability of the protein in the absence of arabinose to bind tightly and specifically when the separation of its half-sites was increased by 10 or 21 bp. In the presence of arabinose the protein could specifically contact both half-sites of a +10 bp spacing construct but could not contact both in a +21 bp construct. Reduced extensibility of AraC protein in the presence of arabinose provides a simple mechanism for the protein's shift from a non-inducing, DNA looping state to an inducing, non-looping state that contacts two adjacent half-sites at the arapBAD promoter.

AraC Transcription Factor↗

PubMed1991

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.

AraC Transcription Factor↗