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Results for “Operator Regions, Genetic”

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PubMed2002

Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin.

Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.

Bacillus subtilis↗

PubMed2001

Engineered disulfide linking the hinge regions within lactose repressor dimer increases operator affinity, decreases sequence selectivity, and alters allostery.

The hinge domain encompasses amino acids 51-60 of lactose repressor (LacI) and plays an important role in its regulatory interaction with operator DNA. This segment makes both hinge-DNA and hinge-hinge' contacts that are critical to DNA binding. Furthermore, this small region serves as a central element in communicating the allosteric response to inducer. Introducing a disulfide bond between partner hinges within a dimer via the mutation V52C results in a protein that has increased affinity for O(1) operator DNA compared to wild-type LacI and abolishes allosteric response to inducer [Falcon, C. M., Swint-Kruse, L., and Matthews, K. S. (1997) J. Biol. Chem. 272, 26818]. We have established that this high affinity is maintained for the disulfide-linked protein even when symmetry and half-site spacing within the operator region are altered, whereas binding by the reduced protein, as for wild-type LacI, is severely diminished by these alterations. Interestingly, the allosteric response to inducer for V52C-oxidized remains intact for a small group of operator variants. Temperature studies demonstrate that the presence of the disulfide alters the thermodynamics of the protein-DNA interaction, with a DeltaC(p) of significantly smaller magnitude compared to wild-type LacI. The results presented here establish the hinge region as an important element not only for LacI high-affinity operator binding but also for the essential communication between ligand binding domains. Moreover, the results confirm that DNA sequence/conformation can profoundly influence allostery for this prototypic regulatory protein.

Allosteric Regulation↗

PubMed1990

How Trp repressor binds to its operator.

We propose that the generally accepted model of a single Trp repressor dimer binding to a center of symmetry in the natural trp operator (Otwinowski et al., 1988) is wrong. We show here that the Trp repressor binds to a sequence whose center is located four base pairs either to the right or to the left of the central axis of symmetry that was previously identified. We show that: (i) the oligonucleotide used by Otwinowski et al. is not retarded by the Trp repressor in a mobility shift assay under conditions wherein a shorter oligonucleotide carrying our consensus sequence is retarded, (ii) that methylation protection experiments on the full natural operator sequence and the short oligonucleotide protect similar patterns and (iii) that by varying every base in the shorter oligonucleotide, we can demonstrate an optimal sequence for Trp repressor binding.

Bacterial Proteins↗

PubMed1994

Operator-constitutive mutations in a DNA sequence recognized by a yeast homeodomain.

Homeodomain proteins regulate transcription in organisms as diverse as yeasts, mammals and plants, often effecting key decisions in development. Although homeodomains can selectively recognize certain DNA sequences, a question has arisen as to how specific this interaction is and how much it contributes to the ability of these proteins to properly select target genes in the cell. This question is particularly an issue in cases where the homeodomain proteins recognize DNA cooperatively with other DNA-binding proteins. In this paper, we examine the issue of DNA binding specificity for the homeodomain of the yeast alpha 2 protein (which recognizes the a-specific gene operator cooperatively with the MCM1 protein) by examining both in vivo and in vitro the effects of point mutations in its recognition sequence. We found that most changes in the homeodomain recognition sequence produced only small effects on both homeodomain affinity as measured in vitro (with and without the helper protein MCM1) and operator function as determined in vivo. This tolerance for operator mutations illustrates in a systematic way the modest DNA-binding specificity of the alpha 2 homeodomain and contrasts with the behavior of many of the bacterial and phage repressors where single point mutations in the operator can have dramatic effects on affinity. This tolerance for different sequences may arise from the fact that most of the interactions made between the alpha 2 homeodomain and the DNA occur through long amino acid side chains; we suggest that these side chains can reconfigure in order to create surfaces complementary to many different DNA sequences. The relaxed DNA-binding specificity of homeodomain proteins such as alpha 2 may be an important feature that permits new regulatory circuits to evolve rapidly from existing components.

Base Sequence↗

PubMed1994

Quality and position of the three lac operators of E. coli define efficiency of repression.

Repression of the lac promoter may be achieved in two different ways: either by interference with the action of RNA polymerase or by interference with CAP activation. We investigated cooperative repression of the Escherichia coli lac operon by systematic conversion of its three natural operators (O1, O2 and O3) on the chromosome. We find that cooperative repression by tetrameric Lac repressor increases with both quality and proximity of the interacting operators. A short distance of 92 bp allows effective repression by two very weak operators (O3, O3). The cooperativity of lac operators is discussed in terms of a local increase of repressor concentration. This increase in concentration depends on flexible DNA which allows loop formation.

Base Sequence↗

PubMed1994

Cooperative DNA-protein interactions. Effects of changing the spacing between adjacent binding sites.

Cooperative binding of specific DNA-binding proteins plays crucial roles in gene regulatory circuitry, and is a model system for interactions between proteins bound to DNA. We have studied coliphage HK022 repressor, which binds to two adjacent operators with a cooperativity parameter of approximately 2000. We examined the effect of changing the spacing between these two operators on cooperativity and on the conformation of the complex. Maximum cooperativity was seen with the wild-type spacing; considerable cooperativity was retained for most spacing variants, but was abolished when the operators lay on opposite faces of the DNA helix. Most spacing variants conferred changes in the conformation of the DNA-protein complex. Our data indicate that the pairwise cooperativity observed with the wild-type spacing results from a conformation that prevents protein-protein contacts with flanking bound dimers. We conclude that protein-DNA complexes involving the same specific binding sites and the same protein molecules can adopt many different conformations, depending on the spacing between the binding sites. This conclusion may be broadly applicable to protein-DNA interactions in other systems.

Allosteric Regulation↗

PubMed1998

A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli.

Interactions between proteins affect a wide variety of biological processes, such as signal transduction and control of gene expression. In order to facilitate the study of protein-protein interactions we have developed a new method for specifically detecting the heterodimerization of two heterologous proteins in the bacterium Escherichia coli. The assay is based on the simultaneous use of protein fusions with an altered specificity and a wild-type LexA repressor DNA-binding domain. We have tested this system with two well known eukaryotic dimerization domains (the Fos and Jun leucine zippers). The two interacting proteins were, respectively, fused to a wild-type and a mutant LexA DNA-binding domain. Their hetero-association is specifically measured by the transcriptional repression of a reporter gene (lacZ) controlled by a hybrid operator containing a wild-type half-site (CTGT) and a mutated operator half-site (CCGT). The hybrid operator/lacZ construct was integrated into the chromosome of the reporter strain (SU202) to avoid possible artefacts due to variations in plasmid copy number. This method should be particularly useful in those cases where one or both partners are also able to form homodimers, since the assay described here is sensitive only to the formation of heterodimers. Furthermore, this assay gives rise to a screenable red/white phenotype on MacConkey-lactose indicator plates, allowing for a genetic study of the specificity of the interaction.

Bacterial Proteins↗

PubMed1988

LexA repressor induces operator-dependent DNA bending.

LexA, the repressor of the SOS system in Escherichia coli induces a substantial DNA bending upon interaction with the operator of the caa gene, which codes for the bacterial toxin colicin A. Analysis by gel electrophoresis of a family of DNA fragments of identical length, but bearing the caa operator at different positions, shows that DNA bending occurs close to or within the operator sequence upon LexA binding. In contrast, the interaction of LexA with the recA operator induces no detectable bending on 5% polyacrylamide gels. This difference between the two operators is likely to be due to an intrinsic bendability of the caa operator related to thymine tracts located on both sides of the operator. Such tracts do not exist in the recA operator. The free DNA fragments harbouring the caa operator show a slight tendency to bend even in the absence of the LexA repressor. The centre of this intrinsic bend is located close to or within the caa operator.

Bacterial Proteins↗

PubMed1992

Interlocking of plasmid DNAs due to Lac repressor-operator interaction.

The presence of a single lac repressor binding sequence on plasmid DNAs is shown to mediate the formation of interlocked dimers in E. coli. The presence of both homo- and hetero-interlocked dimers suggests that the lac repressor complex can bring together randomly two plasmid DNA molecules to facilitate gyrase-mediated interlocking. The exclusive formation of multiply intertwined dimers also suggest that the lac repressor complex may bind simultaneously to a pair of replicated daughter plasmid molecules prior to their segregation. The formation of interlocked plasmid DNAs can be indicative of interaction between two DNA bound proteins in vivo.

Base Sequence↗

PubMed2003

Predicting bacterial transcription units using sequence and expression data.

MOTIVATION: A key aspect of elucidating gene regulation in bacterial genomes is identifying the basic units of transcription. We present a method, based on probabilistic language models, that we apply to predict operons, promoters and terminators in the genome of Escherichia coli K-12. Our approach has two key properties: (i) it provides a coherent set of predictions for related regulatory elements of various types and (ii) it takes advantage of both DNA sequence and gene expression data, including expression measurements from inter-genic probes. RESULTS: Our experimental results show that we are able to predict operons and localize promoters and terminators with high accuracy. Moreover, our models that use both sequence and expression data are more accurate than those that use only one of these two data sources.

Algorithms↗

PubMed1994

Iron-responsive genetic regulation in Campylobacter jejuni: cloning and characterization of a fur homolog.

The Fur protein of Escherichia coli represses transcription from Fur-responsive genes in an iron-dependent manner. We have demonstrated a Fur-like iron-responsive genetic regulatory activity operating in Campylobacter jejuni by using a chloramphenicol acetyl transferase reporter gene separated from its promoter by a synthetic Fur-responsive operator. A fur-like gene has been cloned from C. jejuni by partial functional complementation of an E. coli fur mutation. Sequence analysis has shown that, at the amino acid level, the C. jejuni Fur protein is 35% identical with its E. coli counterpart.

Amino Acid Sequence↗

PubMed1998

Inducible gene expression from African swine fever virus recombinants: analysis of the major capsid protein p72.

A method to study the function of individual African swine fever virus (ASFV) gene products utilizing the Escherichia coli lac repressor-operator system has been developed. Recombinant viruses containing both the lacI gene encoding the lac repressor and a strong virus late promoter modified by the insertion of one or two copies of the lac operator sequence at various positions were constructed. The ability of each modified promoter to regulate expression of the firefly luciferase gene was assayed in the presence and in the absence of the inducer isopropyl beta-D-thiogalactoside (IPTG). Induction and repression of gene activity were dependent on the position(s) of the operator(s) with respect to the promoter and on the number of operators inserted. The ability of this system to regulate the expression of ASFV genes was analyzed by constructing a recombinant virus inducibly expressing the major capsid protein p72. Electron microscopy analysis revealed that under nonpermissive conditions, electron-dense membrane-like structures accumulated in the viral factories and capsid formation was inhibited. Induction of p72 expression allowed the progressive building of the capsid on these structures, leading to assembly of ASFV particles. The results of this report demonstrate that the transferred inducible expression system is a powerful tool for analyzing the function of ASFV genes.

African Swine Fever Virus↗

PubMed1994

A thermodynamic study of the trp repressor-operator interaction.

We have measured the heats of formation of the trp repressor/operator complex by direct titration calorimetry over the temperature range 10 degrees C to 40 degrees C. A primary strong mode of binding displays the characteristic large negative heat capacity change observed by other methods in the formation of specific protein/DNA complexes. Unlike most such reactions, however, the formation of the trp repressor/operator complex is enthalpically driven throughout the physiological temperature range. After saturation of this principal mode, we also detected a secondary weaker binding mode, which we ascribe to a now well documented interaction called "half-site" binding. Although weak, this mode also exhibits an unusually large negative heat capacity change. Since the interface of the proposed secondary half-site binding mode has the same complementary stereochemistry as the primary one (due to internal symmetry), we correlate the negative heat capacity change with the formation of a stereospecific interface and not with high affinity. As in similar cases, the empirical correlation between buried non-polar surfaces and reduction of heat capacity does not account for the large negative delta Cp, nor do crystal structures reveal any further reduction in solvent excluded surfaces within the reactants upon complex formation. We attribute the "unaccounted for" decrement in the heat capacity of the complex to the stereospecific restriction of the hydrated polar elements that form the specific interface. We suggest that the "tightening of soft internal modes" at and near the polar interface of the complex is more important than previously recognized because previous considerations did not take into account the highly hydrated nature of these polar elements and the concomitant reduction in the degrees of freedom of the water structure.

Bacterial Proteins↗

PubMed1994

Identification of high affinity binding sites for LexA which define new DNA damage-inducible genes in Escherichia coli.

A multi-step screening procedure was devised to identify new operators for the LexA repressor in the sequenced portions of the genomes of Escherichia coli and its plasmids and bacteriophages. Sequence analysis methods were employed initially to distinguish true LexA operators from "operator-like" sequences stored within the GenBank and EMBL databases. The affinity of purified LexA protein for cloned DNA fragments containing several of the prospective new sites was then assessed using quantitative electrophoretic mobility shift assays and site-directed mutagenesis. Calculated binding affinities were compared directly with values determined for known and mutant LexA operators in concurrent experiments. Three E. coli chromosomal segments (near pyrC, hsdS and ntrla) and two bacteriophage sequences (near the P1 cre and lambda oop genes) bound LexA protein specifically. These sites and most others identified in the screening are located immediately upstream of known genes and/or large open reading frames. These results and additional transcription data demonstrate that several of the sequences define new DNA damage-inducible (din) genes and include the previously uncharacterized dinD locus. Furthermore, the search identified an SOS gene within the genome of P1 which encodes a protein that is homologous to UmuD', the RecA-promoted cleavage product of the umuD gene. The success of the combinatorial approach described here suggests that analogous searches for new regulatory sequences within the E. coli genome and the genomes of other organisms will also yield favorable results.

Amino Acid Sequence↗

PubMed1993

An engineered PGK promoter and lac operator-repressor system for the regulation of gene expression in mammalian cells.

Previous reports have demonstrated that the Escherichia coli lac repressor can operate effectively in mammalian cells to repress expression of genes driven by modified viral or metallothionein (MT) promoters. We have developed a more general expression system using the promoter from the PGK1 gene (encoding murine 3-phosphoglycerate kinase) which is widely expressed in almost all cell types, including early embryonic and ES (embryonic stem) cells. Firstly, we engineered the lac repressor to include a nuclear localisation signal and placed it under control of the PGK1 promoter. Efficient nuclear localisation of the repressor was demonstrated by mobility-shift assays and immunofluorescence detection. For the target vectors, we modified the wild-type (wt) PGK1 promoter to include lac operator (lacO) sites for binding of the lac repressor and compared a number of different lacO positions and arrangements based on proximity to the native start points for transcription (tsp) and translation. In the absence of repressor, we observed reduced expression of the neo reporter gene for some placements of the lacO, but wt expression for placements near the tsp. When both target and repressor were present in the cells, we observed that the expression of neo could be strongly suppressed and reversibly regulated by induction with IPTG. In particular, for a promoter which contained two spaced lacO replacing native sequence around the major tsp, we observed 90-95% repression by the lac repressor for the neo reporter gene and up to 98% repression for the cat reporter gene. Efficient derepression by IPTG was observed in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2004

Viral evolution as a tool to improve the tetracycline-regulated gene expression system.

We present viral evolution as a novel and powerful method to optimize non-viral proteins. We used this approach to optimize the tetracycline (Tc)-regulated gene expression system (Tet system) for its function in mammalian cells. The components of the Tet system were incorporated in the human immunodeficiency virus (HIV)-1 virus such that viral replication is controlled by this regulatory system. Upon long term replication of this HIV-rtTA virus in human T cells, we obtained a virus variant with an enhanced replication potential resulting from an improved rtTA component of the introduced Tet system. We identified a single amino acid exchange, F86Y, which enhances the transcriptional activity and doxycycline (dox) sensitivity of rtTA. We generated a new rtTA variant that is 5-fold more active at high dox levels than the initial rtTA, and 25-fold more sensitive to dox, whereas the background activity in the absence of dox is not increased. This new rtTA variant will be very useful in biological applications that require a more sensitive or active Tet system. Our results demonstrate that the viral evolution strategy can be used to improve the activity of genes by making them an integral and essential part of the virus.

Biotechnology↗

PubMed1998

Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli.

This report describes an Escherichia coli genetic system that permits bacterial genetic methods to be applied to the study of essentially any prokaryotic or eukaryotic site-specific DNA binding protein. It consists of two parts. The first part is a set of tools that facilitate construction of customized E.coli strains bearing single copy lacZYA reporters that are repressed by a specific target protein. The second part is a pair of regulatable protein expression vectors that permit in vivo production of the target protein at levels appropriate for genetic experiments. When expressed in a properly designed reporter strain, the target protein represses the lac genes, resulting in an E.coli phenotype that can be quantitatively measured or exploited in large scale genetic screens or selections. As a result, large plasmid-based libraries of protein genes or pools of mutagenized variants of a given gene may be examined in relatively simple genetic experiments. The strain construction technique is also useful for generating E.coli strains bearing reporters for other types of genetic systems, including repression-based and activation-based systems in which chimeric proteins are used to examine interactions between foreign protein domains.

Base Sequence↗

PubMed1998

A vector for systematic gene inactivation in Bacillus subtilis.

To study the functions of the uncharacterized open reading frames identified in the Bacillus subtilis genome, several vectors were constructed to perform insertional mutagenesis in the chromosome. All the pMUTIN plasmids carry a lacZ reporter gene and an inducible Pspac promoter, which is tightly regulated and can be induced about 1000-fold. The integration of a pMUTIN vector into the target gene has three consequences: (1) the target gene is inactivated; (2) lacZ becomes transcriptionally fused to the gene, allowing its expression pattern to be monitored; (3) the Pspac promoter controls the transcription of downstream genes in an IPTG-dependent fashion. This last feature is important because B. subtilis genes are often organized in operons. The potential polar effects generated by the integration of the vectors can be alleviated by addition of IPTG. Also, conditional mutants of essential genes can be obtained by integrating pMUTIN vectors upstream of the target gene. The vectors are currently being used for systematic inactivation of genes without known function within the B. subtilis European consortium. pMUTIN characteristics and the inactivation of eight genes in the resA-serA region of the chromosome are presented.

Bacillus subtilis↗