Robert Schleif

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.

Regulation of the L-arabinose operon of Escherichia coli.

Over forty years of research on the L-arabinose operon of Escherichia coli have provided insights into the mechanism of positive regulation of gene activity. This research also discovered DNA looping and the mechanism by which the regulatory protein changes its DNA-binding properties in response to the presence of arabinose. As is frequently seen in focused research on biological subjects, the initial studies were primarily genetic. Subsequently, the genetic approaches were augmented by physiological and then biochemical studies. Now biophysical studies are being conducted at the atomic level, but genetics still has a crucial role in the study of this system.

AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action

This review covers the physiological aspects of regulation of the arabinose operon in Escherichia coli and the physical and regulatory properties of the operons controlling gene, araC. It also describes the light switch mechanism as an explanation for many of the proteins properties. Although many thousands of homologs of AraC exist and regulate many diverse operons in response to many different inducers or physiological states, homologs that regulate arabinose-catabolizing genes in response to arabinose were identified. The sequence similarities among them are discussed in light of the known structure of the dimerization and DNA-binding domains of AraC.

Control of production of ribosomal protein

Abstract The ratio, α (rate of synthesis of ribosomal protein/rate of synthesis of total protein) was measured in Escherichia coli B/r with the following results. 1. (1) In balanced exponential growth in succinate minimal medium, α = 0.08 ± 0.01 at 37 °C. 2. (2) In balanced exponential growth in glucose minimal medium, α = 0.15 ± 0.015 at 37 °C. 3. (3) During the transition period after glucose is added to a culture growing exponentially in succinate, α shifts from 0.08 to 0.15 in two to five minutes. 4. (4) α does not vary with time in a synchronized population growing in glucose minimal medium. Measurement of α required a modification of the sucrose gradient ultracentrifuge technique to allow separation of ribosomes from the soluble proteins without loss of the latter. At 37 °C the doubling times in succinate and in glucose are 100 minutes and 50 minutes, respectively. Combining these results with the steady-state values of a and using the known protein content of a ribosome, the average growth rate of α polypeptide is computed to be 13 ± 2 amino acids per second per ribosome in succinate, glucose, or during the transition between the two media.

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 proteins shift from a non‐inducing, DNA looping state to an inducing, non‐looping state that contacts two adjacent half‐sites at the arapBAD promoter.

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-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.

Mutations in LOXHD1, a recessive-deafness locus, cause dominant late-onset Fuchs corneal dystrophy.

Fuchs corneal dystrophy (FCD) is a genetic disorder of the corneal endothelium and is the most common cause of corneal transplantation in the United States. Previously, we mapped a late-onset FCD locus, FCD2, on chromosome 18q. Here, we present next-generation sequencing of all coding exons in the FCD2 critical interval in a multigenerational pedigree in which FCD segregates as an autosomal-dominant trait. We identified a missense change in LOXHD1, a gene causing progressive hearing loss in humans, as the sole variant capable of explaining the phenotype in this pedigree. We observed LOXHD1 mRNA in cultured human corneal endothelial cells, whereas antibody staining of both human and mouse corneas showed staining in the corneal epithelium and endothelium. Corneal sections of the original proband were stained for LOXHD1 and demonstrated a distinct increase in antibody punctate staining in the endothelium and Descemet membrane; punctate staining was absent from both normal corneas and FCD corneas negative for causal LOXHD1 mutations. Subsequent interrogation of a cohort of >200 sporadic affected individuals identified another 15 heterozygous missense mutations that were absent from >800 control chromosomes. Furthermore, in silico analyses predicted that these mutations reside on the surface of the protein and are likely to affect the proteins interface and protein-protein interactions. Finally, expression of the familial LOXHD1 mutant allele as well as two sporadic mutations in cells revealed prominent cytoplasmic aggregates reminiscent of the corneal phenotype. All together, our data implicate rare alleles in LOXHD1 in the pathogenesis of FCD and highlight how different mutations in the same locus can potentially produce diverse phenotypes.

An l-arabinose binding protein and arabinose permeation in Escherichia coli☆

Abstract A search was made for proteins of the arabinose system which bind arabinose, as it is expected that the permease and possibly the C protein could be identified by arabinose binding. One protein was found and this is most probably part of the arabinose permease. It is inducible, can be removed from cells by osmotic shock and easily purified, and consists of one polypeptide of molecular weight 35,000. In these respects it is closely similar to proteins binding sulfate, leucine, and galactose (Pardee, Prestidge, Whipple & Dreyfuss, 1966; Anraku, 1968; Penrose, Nichoalds, Piperno & Oxender, 1968). Mutants were found with reduced ability to concentrate arabinose intracellularly but which still possessed the arabinose binding proteins. One mutant was found with a lowered level of binding protein. This strain is unable to concentrate any detectable amount of arabinose. None of the mutations affecting permeation of arabinose mapped in the threonine-leucine region where the arabinose genes A , B , C and D are located.

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.