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R F Schleif

Publications and source records attributed to R F Schleif.

At least 37 records · Page 2Linked to original sources
PubMed1990

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.

AraC Transcription Factor↗
PubMed1990

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.

Cell Line↗
PubMed1990

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.

Base Sequence↗
PubMed1989

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.

AraC Transcription Factor↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1987

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.

AraC Transcription Factor↗
PubMed1987

Missing contact probing of DNA-protein interactions.

We have examined the positions of contact between lambda phage repressor protein and operator OR1 DNA by scanning populations of lightly depurinated or depyrimidated DNA for bases essential to or irrelevant to repressor binding. This global scanning technique delineates the apparent contact region between lambda repressor and operator and shows bases previously demonstrated or predicted to be contacted plus some additional bases. A mutant repressor, previously shown to contact DNA as wild-type repressor does with the exception of a missing contact to guanosine G4' [Hochschild, A. & Ptashne, M. (1986) Cell 44, 925-933], similarly failed to contact G4' when assayed by this method. Coupled with altering a test residue of a DNA-contacting protein to glycine or alanine so as to eliminate a specific contact, the method appears to provide an efficient means of scanning for specific residue-base contacts.

Bacteriophage lambda↗
PubMed1987

Cross-induction of the L-fucose system by L-rhamnose in Escherichia coli.

Dissimilation of L-fucose as a carbon and energy source by Escherichia coli involves a permease, an isomerase, a kinase, and an aldolase encoded by the fuc regulon at minute 60.2. Utilization of L-rhamnose involves a similar set of proteins encoded by the rha operon at minute 87.7. Both pathways lead to the formation of L-lactaldehyde and dihydroxyacetone phosphate. A common NAD-linked oxidoreductase encoded by fucO serves to reduce L-lactaldehyde to L-1,2-propanediol under anaerobic growth conditions, irrespective of whether the aldehyde is derived from fucose or rhamnose. In this study it was shown that anaerobic growth on rhamnose induces expression of not only the fucO gene but also the entire fuc regulon. Rhamnose is unable to induce the fuc genes in mutants defective in rhaA (encoding L-rhamnose isomerase), rhaB (encoding L-rhamnulose kinase), rhaD (encoding L-rhamnulose 1-phosphate aldolase), rhaR (encoding the positive regulator for the rha structural genes), or fucR (encoding the positive for the fuc regulon). Thus, cross-induction of the L-fucose enzymes by rhamnose requires formation of L-lactaldehyde; either the aldehyde itself or the L-fuculose 1-phosphate (known to be an effector) formed from it then interacts with the fucR-encoded protein to induce the fuc regulon.

Alcohol Oxidoreductases↗
PubMed1986

The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites.

Two sets of experiments have been performed to test the DNA loop model of repression of the araBAD operon of Escherichia coli. First, dimethyl sulfate methylation protection measurements on normally growing cells show that the AraC regulatory protein occupies the araI site in the presence and absence of the inducer arabinose. Similarly, the araO2 site is shown to be occupied by AraC protein in the presence and absence of arabinose; however, its occupancy by AraC is greatly reduced when araI and adjacent sequences are deleted. Thus, AraC protein binds to araO2 cooperatively with some other component of the ara system located at least 60 base pairs away. Second, the mutational analysis presented here shows that the DNA components required for repression of araBAD are araI, araO2, and perhaps the araBAD operon RNA polymerase binding site.

AraC Transcription Factor↗
PubMed1984

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.

AraC Transcription Factor↗
PubMed1984

An operator at -280 base pairs that is required for repression of araBAD operon promoter: addition of DNA helical turns between the operator and promoter cyclically hinders repression.

A site has been found that is required for repression of the Escherichia coli araBAD operon. This site was detected by the in vivo properties of deletion mutants. In vitro protection studies with DNase I and dimethylsulfate showed that araC protein can specifically bind in this area to nucleotides lying at position -265 to -294 with respect to the araBAD operon promoter (PBAD) transcription start point. The previously known sites of protein binding in the ara operon lie between +20 and -160. Since the properties of deletion strains show that all the sites required for araBAD induction lie between +20 and -110, the new site at -280 exerts its repressive action over an unusually large distance along the DNA. Insertions of -16, -8, 0, 5, 11, 15, 24, and 31 base pairs of DNA between the new site and PBAD were constructed. Repression was impaired in those cases in which half-integral turns of the DNA helix were introduced, but repression was nearly normal for the insertions of 0, +11, and +31 base pairs.

AraC Transcription Factor↗
PubMed1983

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.

AraC Transcription Factor↗
PubMed1982

Hyperproduction of araC protein from Escherichia coli.

Hypersynthesis of araC protein from Escherichia coli has been accomplished. The araC gene was cloned on plasmid pBR322, and some of the noncoding DNA preceding the araC gene was removed by exonuclease digestion. Finally, a DNA fragment containing the lac promoter and ribosome binding site was placed in front the araC gene. By these means the level of araC protein was increased about 5000-fold above the levels found in wild-type cells. This level of protein permits straight forward purification of sizeable quantities of araC protein.

AraC Transcription Factor↗
PubMed1978

In vivo titration of araC protein.

The requirement for araC protein in the induction of the araBAD operon was investigated. Strains of Escherichia coli carrying an araC(Am) mutation and temperature-sensitive amber suppressors were used to vary the intracellular level of araC protein. The levels of araC protein studied ranged from 0.007 to 1.8 times the normal amount. The results indicate that the normal level of araC protein is just sufficient to provide maximal expression of the araBAD operon.

Arabinose↗
PubMed1975

Kinetics of the onset of catabolite repression in Escherichia coli as determined by lac messenger ribonucleic acid initiations and intracellular cyclic adenosine 3',5'-monophosphate levels.

The rates of synthesis of beta-galactosidase (EC 3.2.1.23) and the intracellular levels of cyclic 3',5'-adenosine monophosphate (cAMP) soon after the addition of glucose or glycerol to exponentially growing cultures of Escherichia coli have been determined. Within 10 s of its addition, glucose, but not glycerol, lowered the apparent initiation frequency of lac messenger ribonucleic acid. The glucose-generated reduction in initiations is identified as catabolite repression by its reversibility with cAMP. The intracellular cAMP levels respond virtually identically to glucose and glycerol additions. Thus, no correlation was observed between the rate of messenger ribonucleic acid initiation and the level of cAMP.

Cyclic AMP↗