Biomedical subjects

R Schleif

Publications and source records attributed to R Schleif.

At least 37 records · Page 2Linked to original sources

PubMed1990

Characterization of the Escherichia coli araFGH and araJ promoters.

The identities of two cloned, arabinose-inducible promoters were tested by hybridizing promoter DNA fragments with restriction digests of chromosomal DNA containing Mudlac phage inserted in either araFGH or in araE transport operons. One promoter, thought to be araE, is within 10(3) base-pairs of a Mudlac insertion in the araE gene. The second promoter was not found within several thousand base-pairs of either of the known transport genes. This promoter is now named araPJ (araJ). The DNA sequence of the fragment containing the araFGH promoter was determined. The start site of transcription in vivo was located to within +/- 1 base-pair (bp) by S1 nuclease mapping. DNase 1 footprinting revealed that, in comparison with the araBAD and araE promoters, the locations of the AraC and cyclic AMP receptor protein (CRP) binding sites are reversed with CRP lying between AraC and RNA polymerase. The central location of the CRP binding site may explain why the araFGH promoter is more catabolite sensitive than the other ara promoters. AraC and CRP were both required for maximal transcription in vitro, although a low level of transcription was detected with CRP alone. S1 nuclease mapping of mRNA-DNA hybrids from the araJ promoter located the transcription start point to within #/- 3 bp, and demonstrates that the promoter is dependent upon AraC protein and CRP in vivo. DNase footprinting showed that the location of the AraC protein binding site on araJ is adjacent to the RNA polymerase site, as seen at the araBAD and araE promoters. Two CRP sites were observed; one is upstream from the AraC site and one is downstream from the transcription start site.

Amino Acid Sequence↗

PubMed1989

Determining residue-base interactions between AraC protein and araI DNA.

Depurination/depyrimidation binding-interference experiments (missing contact probing) identified specific candidate residue-base interactions lost by mutants of Escherichia coli L-arabinose operon regulatory protein, AraC, to one of its binding sites, araI. These candidates were then checked more rigorously by comparing the affinities of wild-type and alanine-substituted AraC protein to variants of araI with alterations in the candidate contacted positions. Residues 208 and 212 apparently contact DNA and support, but do not prove the existence of a helix-turn-helix structure in this region of AraC protein whereas contacts by mutants with alterations at positions 256, 257 and 261 which are within another potential helix-turn-helix region do not support the existence of such a structure there. The missing contacts displayed by three AraC mutants are found within two major groove regions of the DNA and are spaced 21 base-pairs apart in a pattern indicating a direct repeat orientation for the subunits of AraC.

Binding Sites↗

PubMed1989

Transvaginal hysterosalpingo-contrast-sonography (Hy-Co-Sy) compared with conventional tubal diagnostics.

Transvaginal hysterosalpingo-contrast-sonography (Hy-Co-Sy) is described as a new method for direct imaging of the tubal passage in tubal diagnostics. During one period of anaesthesia, a transvaginal Hy-Co-Sy was followed by either hysterosalpingography or chromolaparoscopy in 42 patients with sterility disorders. The contrast-enhanced sonography of the tubes was performed in eight cases with sterile saline solution (group 1) and 34 cases with a specially developed ultrasound contrast medium (SH U 454, group 2). The findings obtained by transvaginal Hy-Co-Sy were compared with those of the conventional method. By both methods, there was complete agreement with respect to the same evaluation of tubal passage for both sides, partial agreement when firstly the tubes were assessed as unilaterally or bilaterally open--without precise localization of the side--depending on the appearance of fluid in the pouch of Douglas, or secondly, only one side, when compared, showed agreement. In group 1 complete agreement was found once, partial agreement five times and non-agreement twice, while in group 2 complete agreement was found 22 times (65%), partial agreement 11 times and non-agreement once.

Adult↗

PubMed1988

DNA binding by proteins.

Study of proteins that recognize specific DNA sequences has yielded much information, but the field is still in its infancy. Already two major structural motifs have been discovered, the helix-turn-helix and zinc finger, and numerous examples of DNA-binding proteins containing either of them are known. The restriction enzyme Eco RI uses yet a different motif. Additional motifs are likely to be found as well. There is a growing understanding of some of the physical chemistry involved in protein-DNA binding, but much remains to be learned before it becomes possible to engineer a protein that binds to a specific DNA sequence.

Amino Acids↗

PubMed1988

DNA looping.

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DNA↗

PubMed1988

Alternative DNA loops regulate the arabinose operon in Escherichia coli.

The araCBAD regulatory region of Escherichia coli contains two divergently oriented promoters and three sites to which AraC, the regulatory protein of the operon, can bind. This paper presents the results of in vivo dimethyl sulfate "footprinting" experiments to monitor occupancy of the three AraC sites and measurements of activity of the two promoters. These measurements were made both in the absence of the inducer arabinose and at various times after arabinose addition to growing cells containing the wild-type ara regulatory region or the regulatory region containing various deletions and point mutations. The data lead to the conclusion that two different DNA loops can form in the ara regulatory region. These loops are generated by AraC protein molecules binding to two different DNA sites and binding to each other. One of these loops predominates in the absence of arabinose and plays a major role in repressing activity of one of the promoters. Upon the addition of arabinose the amount of the first loop type, the repression loop, decreases and the amount of a second loop increases. Formation of this second loop precludes the counterproductive formation of the repression loop.

AraC Transcription Factor↗

PubMed1986

Transcription of Escherichia coli ara in vitro. The cyclic AMP receptor protein requirement for PBAD induction that depends on the presence and orientation of the araO2 site.

The mechanism by which the cyclic AMP receptor protein, CRP, stimulates transcription of the Escherichia coli araBAD promoter was studied in vitro. Under one set of conditions, CRP stimulated by eightfold the rate of RNA polymerase open complex formation on supercoiled DNA template containing the normal wild-type araBAD regulatory region. Since previous studies in vivo had identified an upstream site termed araO2 that is involved in both repression and in the CRP requirement for PBAD induction, we performed similar experiments in vitro. Deletion of araO2 or alterations of its orientation with respect to the araI site by half integral numbers of turns greatly reduced the CRP requirement for induction of PBAD. Linearizing the DNA has the same effect as deleting araO2 from the supercoiled DNA template. The similarity of conditions that relieve the classical repression of PBAD in vivo and the conditions that eliminate the requirement for CRP for maximal activity in vitro suggest a close relationship between repression in the ara system and the role of CRP. At lower concentrations of AraC protein and slightly different conditions than those used in the above-mentioned experiments, CRP does stimulate transcription from linear or supercoiled templates lacking araO2. On linear DNA under these conditions, one dimer of AraC protein binds to linear araPBAD DNA, but is incapable of stimulating transcription without the additional binding of CRP. The responses of the ara system under the second set of conditions are unlike its behavior in vivo.

AraC Transcription Factor↗

PubMed1985

Altered DNA contacts made by a mutant AraC protein.

Mutant AraC proteins were selected for their ability to induce but not to repress, or their ability to repress but not to induce the araBAD operon. One such unusual mutant is able to bind to the araI site with an affinity only two to three-fold weaker than the wild type AraC protein, but the mutant protein was shown, both in crude extracts and when purified, to contact only two of the three major groove regions of the DNA that are contacted by the wild type protein.

AraC Transcription Factor↗

PubMed1985

A dimer of AraC protein contacts three adjacent major groove regions of the araI DNA site.

Contact sites of AraC protein to the regulatory site araI of the Escherichia coli araBAD operon have been determined by the chemical-interference technique. DNA fragments were chemically modified an average of once per molecule, and fragments that no longer bound AraC were separated by gel electrophoresis from the DNA fragments still able to bind the protein. The contact sites were then determined by comparing the positions of modifications in the two DNA samples. Strong contacts were found with guanines in three consecutive major groove regions and the adjacent phosphates along one side of the DNA. The conserved bases of the AraC-binding DNA consensus sequence are also found in the same positions. The gel electrophoresis assay was used to determine the stoichiometry of binding, and AraC protein was found to bind the araI and araO1 regulatory sites as a dimer. Therefore, AraC appears to bind DNA differently from the other well-characterized regulatory proteins such as phage lambda repressor.

Alkylation↗

PubMed1984

Upstream repression and CRP stimulation of the Escherichia coli L-arabinose operon.

Repression of the Escherichia coli araBAD promoter, PBAD, was studied using a mutant PBAD promoter (cip-5) that is expressed in the absence of the two proteins required for PBAD induction, AraC protein and the cyclic AMP receptor protein (CRP-cAMP). Like the wild type promoter, cip-5 was repressed by AraC protein, and this repression required a site well upstream of the transcriptional start site. cip-5 was used to determine whether repression results from interference with the functioning of either AraC protein at araI and/or CRP-cAMP. Repression of cip-5 was eliminated by a point mutation within the AraC protein binding site araI but was not affected in the absence of CRP-cAMP. These results suggest that repression involves an interaction between two AraC protein binding sites located over 200 nucleotides apart. Our results also suggest that the majority of the CRP requirement for PBAD is a result of PBAD repression. When repression was abolished by deletion of the araO2 site, the requirement for CRP-cAMP in PBAD induction was greatly reduced.

AraC Transcription Factor↗

PubMed1984

Deletion analysis of the Escherichia coli ara PC and PBAD promoters.

Deletions extending various distances into the ara PC-PBAD regulatory region were studied to define the sites required in vivo for the activity of these promoters. Deletions from the PC side entering the CRP site, which is located from -80 to -120 with respect to the PBAD transcription start site, reduced activity of this promoter. Similarly, deletions entering this site from the PBAD side reduced activity of the PC promoter. Cyclic AMP receptor protein bound at this site apparently functions to stimulate transcription of both flanking promoters.

AraC Transcription Factor↗

PubMed1983

The araE low affinity L-arabinose transport promoter. Cloning, sequence, transcription start site and DNA binding sites of regulatory proteins.

The promoter for the gene encoding the low affinity L-arabinose uptake protein in Escherichia coli was studied. The promoter was cloned, sequenced, its transcription start site determined by S1 nuclease mapping, the proteins required for in vitro transcription were determined, and the regulatory protein binding sites located by DNase footprinting. The araE promoter shows no evidence of an operator site upstream from the CRP binding site, but otherwise it is similar to the araBAD promoter.

Arabinose↗

PubMed1983

Spacing mutations between the Escherichia coli pBAD RNA polymerase binding site and the araC (I) induction site.

Mutations in the Escherichia coli promoter PBAD have been constructed which alter the spacing of the adjacent RNA polymerase and araC inducer protein binding sites. While deletion of a single base-pair or small insertions do not detectably affect araC protein binding to DNA and they do not alter the conserved sequence of the RNA polymerase binding site, stimulation of PBAD in vivo is greatly reduced. The experiments suggest that the distance or angle between the two proteins on the DNA is critical for promoter function.

AraC Transcription Factor↗

PubMed1983

In vivo regulation of the Escherichia coli araC promoter.

The ara pC promoter is known to be derepressed about fivefold for 20 to 30 min after the addition of arabinose. This transient derepression was studied by using araC::Mu lac insertions and araC-lacZ gene fusions. In strains containing increased levels of araC protein, the pC promoter became progressively less derepressible, but the ara pBAD promoter remained normally inducible. Repression of pC was reestablished 20 min after induction in araB mutants, but did not occur in arabinose-transport-deficient mutants. Finally, mutant araCc proteins which normally do not repress pC did so in the presence of arabinose.

Arabinose↗

PubMed1982