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R Schleif

Publications and source records attributed to R Schleif.

At least 19 recordsLinked to original sources
PubMed2001

Mapping arm-DNA-binding domain interactions in AraC.

AraC protein, the regulator of the l-arabinose operon in Escherichia coli has been postulated to function by a light switch mechanism. According to this mechanism, it should be possible to find mutations in the DNA-binding domain of AraC that result in weaker arm-DNA-binding domain interactions and which make the protein constitutive, that is, it no longer requires arabinose to activate transcription. We isolated such mutations by randomizing three contiguous leucine residues in the DNA-binding domain, and then by systematically scanning surface residues of the DNA-binding domain with alanine and glutamic acid. As a result, a total of 20 constitutive mutations were found at ten different positions. They form a contiguous trail on the DNA-distal face of the DNA-binding domain, and likely define the region where the N-terminal arm that extends from the N-terminal dimerization domain contacts the C-terminal DNA-binding domain.

Alanine↗
PubMed2001

Identification of oligomerizing peptides.

The AraC DNA binding domain is inactive in a monomeric form but can activate transcription from the arabinose operon promoters upon its dimerization. We used this property to identify plasmids encoding peptide additions to the AraC DNA binding domain that could dimerize the domain. We generated a high diversity library of plasmids by inserting 90-base oligonucleotides of random sequence ahead of DNA coding for the AraC DNA binding domain in an expression vector, transforming, and selecting colonies containing functional oligomeric peptide-AraC DNA binding domain chimeric proteins by their growth on minimal arabinose medium. Six of seven Ara(+) candidates were partially characterized, and one was purified. Equilibrium analytical centrifugation experiments showed that it dimerizes with a dissociation constant of approximately 2 micrometer.

Amino Acid Sequence↗
PubMed2001

The role of rigidity in DNA looping-unlooping by AraC.

We applied two experiments useful in the study of ligand-regulated DNA binding proteins to AraC, the dimeric regulator of the Escherichia coli l-arabinose operon. In the absence of arabinose, AraC prefers to loop DNA by binding to two half-sites that are separated by 210 base pairs, and in the presence of arabinose it prefers to bind to adjacently located half-sites. The basis for this ligand-regulated shift in binding appears to result from a shift in the rigidity of the system, where rigidity both in AraC protein in the absence of arabinose, and in the DNA are required to generate the free energy differences that produce the binding preferences. Eliminating the dimerization domains and connecting the two DNA binding domains of AraC by a flexible peptide linker should provide a protein whose behavior mimics that of AraC when there is no interaction between its dimerization and DNA binding domains. The resulting protein bound to adjacent half-sites on the DNA, like AraC protein in the presence of arabinose. When the two double-stranded DNA half-sites were connected by 24 bases of single-stranded, flexible DNA, wild-type AraC protein bound to the DNA in the presence and absence of arabinose with equal affinity, showing that AraC modulates its DNA binding affinity in response to arabinose by shifting the relative positions of its DNA binding domains. These results are consistent with the light switch mechanism for the action of AraC, refine the model, and extend the range of experimental tests to which it has been subjected.

Amino Acid Sequence↗
PubMed2000

The C-terminal end of AraC tightly binds to the rest of its domain.

Genes were synthesized to express two DNA binding domains of AraC connected by short linkers. The abilities of the resulting proteins to bind to DNA containing AraC half-sites separated by the usual four bases as well as an additional two or three helical turns of the DNA were measured. The inability of some of the protein constructs to bind to widely separated half-sites indicates that the C-terminal 14 amino acids of AraC are firmly bound to the rest of the DNA binding domain.

Amino Acid Sequence↗
PubMed2000

Strengthened arm-dimerization domain interactions in AraC.

Constitutive mutations were sought and found in the N-terminal arm of the Escherichia coli regulatory protein of the arabinose operon, AraC protein. A new mutation, N16D, was of particular interest. Asn-16 is not seen in the crystal structure of the AraC dimerization domain determined in the absence of arabinose, because the N-terminal arm 18 residues are disordered, but in the presence of arabinose, residues 7-18 fold over the arabinose and make many interactions with it. In this state Asn-16 lies near two positively charged amino acids, Lys-43 and Arg-99. We propose that the introduction of the negatively charged aspartic residue at position 16 creates a charge-charge interaction network among Asp-16, Lys-43, and Arg-99 that holds the arm to the dimerization domain even in the absence of arabinose. This frees the DNA-binding domains and allows them to bind cis to I(1)-I(2) half-sites and activate transcription. Mutating the two positively charged residues to alanines individually and collectively decreased or eliminated the constitutivity created by the N16D mutation.

AraC Transcription Factor↗
PubMed2000

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.

Arabinose↗
PubMed2000

Recognition of overlapping nucleotides by AraC and the sigma subunit of RNA polymerase.

The Escherichia coli promoter p(BAD), under the control of the AraC protein, drives the expression of mRNA encoding the AraB, AraA, and AraD gene products of the arabinose operon. The binding site of AraC at p(BAD) overlaps the RNA polymerase -35 recognition region by 4 bases, leaving 2 bases of the region not contacted by AraC. This overlap raises the question of whether AraC substitutes for the sigma subunit of RNA polymerase in recognition of the -35 region or whether both AraC and sigma make important contacts with the DNA in the -35 region. If sigma does not contact DNA near the -35 region, p(BAD) activity should be independent of the identity of the bases in the hexamer region that are not contacted by AraC. We have examined this issue in the p(BAD) promoter and in a second promoter where the AraC binding site overlaps the -35 region by only 2 bases. In both cases promoter activity is sensitive to changes in bases not contacted by AraC, showing that despite the overlap, sigma does read DNA in the -35 region. Since sigma and AraC are thus closely positioned at p(BAD), it is possible that AraC and sigma contact one another during transcription initiation. DNA migration retardation assays, however, showed that there exists only a slight degree of DNA binding cooperativity between AraC and sigma, thus suggesting either that the normal interactions between AraC and sigma are weak or that the presence of the entire RNA polymerase is necessary for significant interaction.

AraC Transcription Factor↗
PubMed1998

Catabolite gene activator protein mutations affecting activity of the araBAD promoter.

We have studied catabolite gene activator protein (CAP) activation at the araBAD promoter, pBAD, in the absence of DNA looping. We ruled out the two most plausible indirect activation mechanisms: CAP-induced folding of upstream DNA back upon RNA polymerase, and CAP-induced stabilization of AraC binding to DNA. Therefore, a direct CAP-RNA polymerase interaction seemed likely. We sought and found CAP mutants defective in transcription activation at pBAD that retained normal DNA binding affinity. Some mutations altered residues in the interval from positions 150 to 164 that includes CAP activating region 1 (AR1), which has been shown to contact RNA polymerase at a number of promoters. Unexpectedly, additional mutations were found that altered residues in the region between positions 46 and 68 and at position 133. This includes the region known as activating region 3 (AR3). Mutations from both groups also affect the araFGH and rhaBAD promoters.

Cyclic AMP Receptor Protein↗
PubMed1997

The 1.6 A crystal structure of the AraC sugar-binding and dimerization domain complexed with D-fucose.

The crystal structure of the sugar-binding and dimerization domain of the Escherichia coli gene regulatory protein, AraC, has been determined in complex with the competitive inhibitor D-fucose at pH 5.5 to a resolution of 1.6 A. An in-depth analysis shows that the structural basis for AraC carbohydrate specificity arises from the precise arrangement of hydrogen bond-forming protein side-chains around the bound sugar molecule. van der Waals interactions also contribute to the epimeric and anomeric selectivity of the protein. The methyl group of D-fucose is accommodated by small side-chain movements in the sugar-binding site that result in a slight distortion in the positioning of the amino-terminal arm. A comparison of this structure with the 1.5 A structure of AraC complexed with L-arabinose at neutral pH surprisingly revealed very small structural changes between the two complexes. Based on solution data, we suspect that the low pH used to crystallize the fucose complex affected the structure, and speculate about the nature of the changes between pH 5.5 and neutral pH and their implications for gene regulation by AraC. A comparison with the structurally unrelated E. coli periplasmic sugar-binding proteins reveals that conserved features of carbohydrate recognition are present, despite a complete lack of structural similarity between the two classes of proteins, suggesting convergent evolution of carbohydrate binding.

Amino Acid Sequence↗
PubMed1997

Structural basis for ligand-regulated oligomerization of AraC.

The crystal structure of the arabinose-binding and dimerization domain of the Escherchia coli gene regulatory protein AraC was determined in the presence and absence of L-arabinose. The 1.5 angstrom structure of the arabinose-bound molecule shows that the protein adopts an unusual fold, binding sugar within a beta barrel and completely burying the arabinose with the amino-terminal arm of the protein. Dimer contacts in the presence of arabinose are mediated by an antiparallel coiled-coil. In the 2.8 angstrom structure of the uncomplexed protein, the amino-terminal arm is disordered, uncovering the sugar-binding pocket and allowing it to serve as an oligomerization interface. The ligand-gated oligomerization as seen in AraC provides the basis of a plausible mechanism for modulating the protein's DNA-looping properties.

AraC Transcription Factor↗
PubMed1997

Arac/XylS family of transcriptional regulators.

The ArC/XylS family of prokaryotic positive transcriptional regulators includes more than 100 proteins and polypeptides derived from open reading frames translated from DNA sequences. Members of this family are widely distributed and have been found in the gamma subgroup of the proteobacteria, low- and high-G + C-content gram-positive bacteria, and cyanobacteria. These proteins are defined by a profile that can be accessed from PROSITE PS01124. Members of the family are about 300 amino acids long and have three main regulatory functions in common: carbon metabolism, stress response, and pathogenesis. Multiple alignments of the proteins of the family define a conserved stretch of 99 amino acids usually located at the C-terminal region of the regulator and connected to a nonconserved region via a linker. The conserved stretch contains all the elements required to bind DNA target sequences and to activate transcription from cognate promoters. Secondary analysis of the conserved region suggests that it contains two potential alpha-helix-turn-alpha-helix DNA binding motifs. The first, and better-fitting motif is supported by biochemical data, whereas existing biochemical data neither support nor refute the proposal that the second region possesses this structure. The phylogenetic relationship suggests that members of the family have recruited the nonconserved domain(s) into a series of existing domains involved in DNA recognition and transcription stimulation and that this recruited domain governs the role that the regulator carries out. For some regulators, it has been demonstrated that the nonconserved region contains the dimerization domain. For the regulators involved in carbon metabolism, the effector binding determinants are also in this region. Most regulators belonging to the AraC/XylS family recognize multiple binding sites in the regulated promoters. One of the motifs usually overlaps or is adjacent to the -35 region of the cognate promoters. Footprinting assays have suggested that these regulators protect a stretch of up to 20 bp in the target promoters, and multiple alignments of binding sites for a number of regulators have shown that the proteins recognize short motifs within the protected region.

Amino Acid Sequence↗
PubMed1996

Obligatory activator-polymerase addition order at promoters.

The kinetics of open complex formation were measured by migration retardation assay and DNase I footprinting at the activator-dependent promoters ara P1, lac P1 and gal P1. In each case, the rate of open complex formation was significantly faster if the activator, AraC for ara and CAP for lac and gal, had been added before RNA polymerase. The results indicate that complexes of transcriptional activators, RNA polymerase and promoter can exist in two states, one which can form open complexes rapidly and one which cannot.

AraC Transcription Factor↗
PubMed1996

Transcription activation parameters at ara pBAD.

We studied the formation of open complexes of RNA polymerase and promoter DNA as activated by the AraC protein at the Escherichia coli araBAD promoter pBAD and by the cyclic AMP receptor protein at the galKTE promoter P1. The DNA migration retardation assay was demonstrated to be suitable for the detection and quantitation of open complexes by the correspondence in the properties of open complexes in solution and retarded complexes observed in gels. These included, on the ara promoter, heparin resistance, lifetime, DNAseI footprinting, exonuclease III footprinting, permanganate footprinting and disappearance upon transcription, and on the gal promoter, the correspondence between the kinetic parameters Kd and k2 obtained with established techniques and those obtained with the migration retardation assay. On the pBAD promoter we obtained kinetic parameters of Kd = 0.3 nM and K2 = 1 minute(-1). The unusually tight binding of polymerase in the presence of AraC suggests that AraC binds polymerase tightly.

AraC Transcription Factor↗
PubMed1993

AraC protein can activate transcription from only one position and when pointed in only one direction.

At the araBAD promoter, the RNA polymerase-proximal half-site for AraC binding partially overlaps the -35 region. Random and explicit spacing experiments show that both this partial overlapping and AraC binding to the polymerase-proximal half-site are necessary and sufficient for strong transcriptional activation. Normally, this occupancy is generated by the presence of arabinose, which shifts AraC from a DNA looping interaction involving the polymerase-distal half-site and the araO2 site 210 base-pairs away, to an interaction with the two half-sites adjacent to RNA polymerase. Changing the polymerase-proximal half-site to a higher affinity AraC binding site gives activation in the absence of arabinose. Thus, arabinose is not required to transform AraC into an activating conformation. Because the two half-sites of araI are direct repeats, the RNA polymerase proximal and distal surfaces of AraC are not identical. When the araI site was turned around, no spacings were found from which AraC could activate transcription. In light of the strict spacing and orientation requirements for AraC activation, the interactions between AraC and RNA polymerase are likely to be specific and inflexible.

AraC Transcription Factor↗
PubMed1992

Repression of the araBAD promoter from araO1.

DNA looping between the araO2 and araI sites holds the uninduced or basal level of expression of the araBAD genes from the pBAD promoter at a low level. Despite the presence of another and closer site potentially capable of mediating looping to araI, no repression from this site, called araO1, is observed. Here we show, using both in vivo and in vitro experiments, that the araO1 site is not normally occupied by AraC protein under repressing conditions, but that if AraC protein is overproduced and the araO2 site is absent, araO1 is then occupied and repression of pBAD can be observed.

AraC Transcription Factor↗
PubMed1992

DNA looping.

DNA looping is widely used in nature. It is well documented in the regulation of prokaryotic and eukaryotic gene expression, DNA replication, and site-specific DNA recombination. Undoubtedly looping also functions in other protein-DNA transactions such as repair and chromosome segregation. While the underlying physical chemistry of DNA looping is common to all systems, the precise biochemical details of looping and the utilization of looping by different systems varies widely. Looping appears to have been chosen by nature in such a wide variety of contexts because it solves problems both of binding and of geometry. The cooperativity inherent in binding a protein to multiple sites on DNA facilitates high occupancy of DNA sites by low concentrations of proteins. DNA looping permits a sizeable number of DNA-binding proteins to interact with one of their number, for example RNA polymerase. Finally, DNA looping may simplify evolution by not requiring a precise spacing between a protein's binding site and a second site on the DNA.

Chemical Phenomena↗
PubMed1991

Mapping, sequence, and apparent lack of function of araJ, a gene of the Escherichia coli arabinose regulon.

We report the mapping, sequencing, and study of the physiological role of the fourth arabinose-inducible operon from Escherichia coli, araJ. It is located at 9 min on the chromosome and codes for a single 42-kDa protein that shows no significant homology to other known proteins. Destruction of the chromosomal araJ gene does not detectably affect either of the two arabinose transport systems, the ability of cells to grow on arabinose, or the induction kinetics of the araBAD operon, and thus the physiological role of AraJ, if any, remains unknown. We have also found a long open reading frame upstream of araJ. The sequence of this upstream open reading frame was found to be identical to the previously reported sequence of the sbcC gene (I. S. Naom, S. J. Morton, D. R. F. Leach, and R. G. Lloyd, Nucleic Acids Res. 17:8033-8044, 1989). The carboxyl region of SbcC has an amino acid sequence consistent with this region of SbcC forming an extended alpha-helical coiled-coil.

Amino Acid Sequence↗