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PubMed2006

Analytical study of the multiplicity of regulatory mechanisms in the tryptophan operon.

In this paper we study the stability of a previously introduced model for the tryptophan operon regulatory pathway. For this, we make use of the second Lyapunov's method. The results obtained for the wild-type and for a couple ofin-silico mutant bacterial strains allow a deeper understanding of the multiplicity of regulatory mechanisms in this operon. In particular, we confirm that enzyme inhibition and transcription attenuation strengthen the system stability, the effect of transcription attenuation being much shorter than that of enzyme inhibition. Furthermore, the analysis here presented provides some insights about how enzyme inhibition affects the system stability.

Algorithms↗
PubMed1986

Interaction of mutant lambda repressors with operator and non-operator DNA.

We have described a set of mutations that alter side-chains on the operator binding surface of lambda repressor. In this paper, we study the interactions of 12 purified mutant repressors with operator and non-operator DNA. The mutant proteins have operator affinities that are reduced from tenfold to greater than 10,000-fold compared to wild-type. Nine of the mutants have affinities for non-operator DNA that are similar to wild-type, two mutants show decreased non-specific binding, and one mutant has increased affinity for non-operator DNA. We discuss these findings in terms of the structural and energetic contributions of side-chain--DNA interactions, and show that certain contacts between the repressor and the operator backbone contribute both energy and specificity to the interaction.

Amino Acid Sequence↗
PubMed2004

Integrative plasmid vector for constructing single-copy reporter systems to study gene regulation in Rhizobium meliloti and related species.

The integrative system of phage 16-3 of Rhizobium meliloti 41 was shown to function in several bacterial species belonging to the Rhizobium, Bradyrhizobium, Azorhizobium, and Agrobacterium genera. It might also function in many other bacterial species provided that both the target site (attB) and the required host factor(s) are present. Here we report on the construction of a new integrative vector that can be utilized in gene regulation studies. It provides an opportunity to create a single-copy set-up for characterizing DNA-protein interactions in vivo, in a wide range of bacteria. To demonstrate the usefulness of the vector, transcription repression by binding of the C repressor protein of phage 16-3 to wild type operators was studied. The assay system provided highly reproducible quantitative data on repression.

Agrobacterium tumefaciens↗
PubMed1999

Criss-crossed interactions between the enhancer and the att sites of phage Mu during DNA transposition.

A bipartite enhancer sequence (composed of the O1 and O2 operator sites) is essential for assembly of the functional tetramer of phage Mu transposase (MuA) on supercoiled DNA substrates. A three-site interaction (LER) between the left (L) and right (R) ends of Mu (att sites) and the enhancer (E) precedes tetramer assembly. We have dissected the role of the enhancer in tetramer assembly by using two transposase proteins that have a common att site specificity, but are distinct in their enhancer specificity. The activity of these proteins on substrates containing hybrid enhancers reveals a 'criss-crossed' pattern of interaction between att and enhancer sites. The left operator, O1, of the enhancer interacts specifically with the transposase subunit at the R1 site (within the right att sequence) that is responsible for cleaving the left end of Mu. The right operator, O2, shows a preferential interaction with the transposase subunit at the L1 site (within the left att sequence) that is responsible for cleaving the right end of Mu.

Attachment Sites, Microbiological↗
PubMed1991

Computer simulations and experimental studies of gel mobility patterns for weak and strong non-cooperative protein binding to two targets on the same DNA: application to binding of tet repressor variants to multiple and single tet operator sites.

A series of computer simulations of gel patterns assuming non-cooperative binding of a protein to two targets on the same DNA fragment was performed and applied to interprete gel mobility shift experiments of Tet repressor-tet operator binding. While a high binding affinity leads to the expected distribution of free DNA, DNA bound by one repressor dimer and DNA bound by two repressor dimers, a lower affinity or an increased electrophoresis time results in the loss of the band corresponding to the singly occupied complex. The doubly occupied complex remains stable under these conditions. This phenomenon is typical for protein binding to DNA fragments with two identical sites. It results from statistical disproportionation of the singly occupied complex in the gel. The lack of the singly occupied complex is commonly taken to indicate cooperative binding, however, our analysis shows clearly, that cooperativity is not needed to interprete these results. Tet repressor proteins and small DNA fragments with two tet operator sites have been prepared from four classes of tetracycline resistance determinants. The results of gel mobility shift analyses of various complexes of these compounds confirm the predictions. Furthermore, calculated gel patterns assuming different gel mobilities of the two singly occupied complexes show discrete bands only if the electrophoresis time is shorter than the inverse of the microscopic dissociation rate constant. Simulations assuming increasing dissociation rates predict that the two bands first merge into one, which then disappears. This behavior was verified by gel mobility analyses of Tet repressor-tet operator titrations at increased salt concentrations as well as by direct footprinting of the complexes in the gel. It is concluded that comparison of the intensities of the single and the double occupation bands allow a rough estimation of the dissociation rate constant. On this basis the sixteen possible Tet repressor-tet operator combinations can be ordered with decreasing binding affinities by a simple gel shift experiment. The implications of these results for gel mobility analyses of other protein-DNA complexes are discussed.

Binding Sites↗
PubMed2006

Evolutionary potential of a duplicated repressor-operator pair: simulating pathways using mutation data.

Ample evidence has accumulated for the evolutionary importance of duplication events. However, little is known about the ensuing step-by-step divergence process and the selective conditions that allow it to progress. Here we present a computational study on the divergence of two repressors after duplication. A central feature of our approach is that intermediate phenotypes can be quantified through the use of in vivo measured repression strengths of Escherichia coli lac mutants. Evolutionary pathways are constructed by multiple rounds of single base pair substitutions and selection for tight and independent binding. Our analysis indicates that when a duplicated repressor co-diverges together with its binding site, the fitness landscape allows funneling to a new regulatory interaction with early increases in fitness. We find that neutral mutations do not play an essential role, which is important for substantial divergence probabilities. By varying the selective pressure we can pinpoint the necessary ingredients for the observed divergence. Our findings underscore the importance of coevolutionary mechanisms in regulatory networks, and should be relevant for the evolution of protein-DNA as well as protein-protein interactions.

Computational Biology↗
PubMed1996

Dual regulation of open-complex formation and promoter clearance by Arc explains a novel repressor to activator switch.

In studies of variants of the P(ant) promoter of bacteriophage P22, the Arc protein was found not only to slow the rate at which RNA polymerase forms open complexes but also to accelerate the rate at which the enzyme clears the promoter. These dual activities permit Arc, bound at a single operator subsite, to act as an activator or as a repressor of different promoter variants. For example, Arc activates a P(ant) variant for which promoter clearance is rate limiting in the presence and absence of Arc but represses a closely related variant for which open-complex formation becomes rate limiting in the presence of Arc. The acceleration of promoter clearance by Arc requires occupancy of the operator subsite proximal to the -35 region and is diminished when Arc bears a mutation in Arg-23, a residue that makes a DNA-backbone contact in the operator complex.

DNA Footprinting↗
PubMed2004

Calculating biological behaviors of epigenetic states in the phage lambda life cycle.

The biology and behavior of bacteriophage lambda regulation have been the focus of classical investigations of molecular control of gene expression. Both qualitative and quantitative aspects of this behavior have been systematically characterized experimentally. Complete understanding of the robustness and stability of the genetic circuitry for the lysis-lysogeny switch remains an unsolved puzzle. It is an excellent test case for our understanding of biological behavior of an integrated network based on its physical, chemical, DNA, protein, and functional properties. We have used a new approach to non-linear dynamics to formulate a new mathematical model, performed a theoretical study on the phage lambda life cycle, and solved the crucial part of this puzzle. We find a good quantitative agreement between the theoretical calculation and published experimental observations in the protein number levels, the lysis frequency in the lysogen culture, and the lysogenization frequency for mutants of O(R). We also predict the desired robustness for the lambda genetic switch. We believe that this is the first successful example in the quantitative calculation of robustness and stability of the phage lambda regulatory network, one of the simplest and most well-studied regulatory systems.

Bacteriophage lambda↗
PubMed1987

Expression of ricin A chain in Escherichia coli.

DNA encoding ricin A chain was derived from preproricin cDNA and ligated into the expression vector pDS5/3. Transcription is controlled from the coliphage promoter PN25 fused with the lac operator of E.coli. When induced, E.coli 71.18 cells transformed with the recombinant plasmid express ricin A chain which is soluble and has full biological activity.

DNA↗
PubMed1997

Complementation of a yeast delta pkc1 mutant by the Arabidopsis proteinANT.

The Saccharomyces cerevisiae protein kinase C homologue, PKC1, is involved in maintenance of cell integrity during polarized growth. We have used a mutant complementation approach to investigate related signal transduction pathways in higher plants. Here we report the isolation of a cDNA from Arabidopsis thaliana which partially suppresses the lytic defect of a delta pkc1 yeast strain. The encoded protein, ANT, belongs to the AP2-related gene family and is essential for ovule development. Expression in yeast of a LexA-ANT fusion protein activates transcription of a reporter gene from promoters containing lexA operators. Our results support the idea that ANT acts as transcriptional activator in planta.

Arabidopsis↗
PubMed2000

A comparative study of the immunity region of lambdoid phages including Shiga-toxin-converting phages: molecular basis for cross immunity.

Comparison of eight lambdoid phages, including three Shiga-toxin converting phages, has been carried out with respect to the immunity region, especially the recognition helices of their repressor and CRO proteins on the one hand, and operator sequences on the other. Some as yet unassigned components of the regulatory circuits have been inferred by computer search. The cross immunity phenomenon shown by phages VT2-Sa and lambda is explained on the basis of similarity in their sequences. In addition, the similarity of 933W and HK022 in the sequences of their recognition helices of repressor and CRO, on the one hand, and operators, on the other, has led us to predict that they will have identical or similar immunity specificity. This homology has enabled us also to locate the OL (and consequently PL) of phage 933W that has been thought to be non-existent.

Amino Acid Motifs↗
PubMed1999

Regulation of pelD and pelE, encoding major alkaline pectate lyases in Erwinia chrysanthemi: involvement of the main transcriptional factors.

The main virulence factors of the phytopathogenic bacterium Erwinia chrysanthemi are pectinases which attack pectin, the major constituent of the plant cell wall. Of these enzymes, the alkaline isoenzyme named PelD in strain 3937 and PelE in strain EC16 has been described as being particularly important, based on virulence studies of plants. Expression of the pelD and pelE genes is tightly modulated by various regulators, including the KdgR repressor and the cyclic AMP-cyclic AMP receptor protein (CRP) activator complex. The use of a lacZ reporter gene allowed us to quantify the repression of E. chrysanthemi 3937 pelD expression exerted by PecS, another repressor of pectinase synthesis. In vitro DNA-protein interaction experiments, centered on the pelD and pelE wild-type or pelE mutated promoter regions, allowed us to define precisely the sequences involved in the binding of these three regulators and of RNA polymerase (RNAP). These studies revealed an unusual binding of the KdgR repressor and suggested the presence of a UP (upstream) element in the pelD and pelE genes. Investigation of the simultaneous binding of CRP, KdgR, PecS, and the RNAP to the regulatory region of the pelD and pelE genes showed that (i) CRP and RNAP bind cooperatively, (ii) PecS partially inhibits binding of the CRP activator and of the CRP-RNAP complex, and (iii) KdgR stabilizes the binding of PecS and prevents transcriptional initiation by RNAP. Taken together, our data suggest that PecS attenuates pelD and pelE expression rather than acting as a true repressor like KdgR. Overall, control of the pelD and pelE genes of E. chrysanthemi appears to be both complex and novel.

Alkalies↗
PubMed1999

Characterization of a manganese-dependent regulatory protein, TroR, from Treponema pallidum.

Genome sequence analysis of Treponema pallidum, the causative agent of syphilis, suggests that this bacterium has a limited iron requirement with few, if any, proteins that require iron. Instead, T. pallidum may use manganese-dependent enzymes for metabolic pathways. This strategy apparently alleviates the necessity of T. pallidum to acquire iron from the host, thus overcoming iron limitation, which is a primary host defense. Interestingly, a putative metal-dependent regulatory protein, TroR, which has homology with the diphtheria toxin regulatory protein, DtxR, from Corynebacterium diphtheriae was identified from T. pallidum. We describe here the characterization of TroR, a regulatory protein. Mobility-shift DNA binding and DNase I footprint assays indicated that purified TroR bound to a 22-nt region of dyad symmetry that overlaps the -10 region of the promoter of the tro operon, which contains the genes for a putative metal transport system, the glycolytic enzyme phosphoglycerate mutase, and TroR. Unlike other metal-dependent regulatory proteins like diphtheria toxin regulatory protein and the ferric ion uptake regulator, Fur, which can be activated by divalent metals such as Fe(2+), Mn(2+), Co(2+), Ni(2+), and Zn(2+), TroR is activated only by Mn(2+). The TroR-Mn(2+) complex binds its target sequence and blocks transcription of the troPO/lacZ fusion, suggesting that TroR acts as a metal-dependent repressor in vivo. In addition, TroR exists as a dimer in both its inactive (metal free) and active states as indicated by chemical crosslinking experiments. Based on these data, we propose that TroR represents a unique regulatory system for controlling gene expression in T. pallidum in response to Mn(2+).

Bacterial Proteins↗
PubMed1988

The Escherichia coli LexA repressor-operator system works in mammalian cells.

We have demonstrated the use of the Escherichia coli LexA repressor-operator system to down-regulate gene expression in mouse cells. The LexA gene was placed downstream of the RSVLTR promoter with polyadenylation and splice signals from SV40. This expression unit was introduced into mouse Ltk- cells by calcium phosphate transfection and stable transfectants selected which express LexA protein. We have used the bacterial chloramphenicol acetyltransferase gene (CAT) as our reporter gene. Transcription of this gene was driven by the HSV tk promoter, into which we have introduced one or two synthetic LexA operator sequences in various positions throughout the promoter. Necessary 3' signals were from the HSV tk gene. Repression by LexA was assessed by comparing the transient expression of tkCAT target constructs, containing LexA operator sequences in the promoter, in cells expressing LexA protein with that in control cells not expressing the repressor. We have observed up to 10-fold repression of CAT expression in LexA+ cells from promoters containing LexA operator sequences.

Animals↗
PubMed2003

Novel macrolide-adjustable bidirectional expression modules for coordinated expression of two different transgenes in mice.

BACKGROUND: Precise control of transgene expression is essential for a variety of applications ranging from gene-function analysis, biopharmaceutical manufacturing to next-generation molecular interventions in gene therapy and tissue engineering. The regulation of gene expression is currently a key issue for clinical implementation of gene-therapy-based treatments since desired transgene expression may need to be maintained within a narrow therapeutic window for successful treatment of a particular human disease. METHODS: We have designed a novel bidirectional expression module that enables adjustable coregulation of two different transgenes in response to clinical doses of macrolide antibiotics. A bidirectional macrolide-responsive promoter consisting of a central operator module (ETR) specific for the macrolide-dependent transactivator (ET1) is flanked by two minimal promoters (P(hCMVmin); P(hsp70min)) which drive expression of two divergently oriented transgenes. Macrolide antibiotics modulate the binding affinity of ET1 to ETR and adjust expression of both transgenes to desired levels. RESULTS: Bidirectional expression configurations enabled excellent macrolide-adjustable coregulation profiles of two secreted reporter genes or one-vector-based autoregulated fine-tuning of a single transgene in various transgenic rodent and human cell lines. Following implantation of microencapsulated CHO-K1 cell derivatives transgenic for macrolide-controlled bidirectional expression of erythropoietin (EPO) and the human secreted alkaline phosphatase (SEAP) intraperitoneally into mice, serum EPO and SEAP levels could be coadjusted to desired levels by administration of different erythromycin doses. CONCLUSIONS: Based on their in vivo compatibility, the versatile bidirectional and macrolide-responsive expression modules represent an important advancement on the way to implementing targeted and conditional molecular interventions into a clinical reality.

Alkaline Phosphatase↗
PubMed1993

Yeast a1 and alpha 2 homeodomain proteins form a DNA-binding activity with properties distinct from those of either protein.

The yeast a1 and alpha 2 proteins are examples of homeodomain proteins that display cell-type-specific expression. They are co-expressed in only one type of cell, the a/alpha cell, where they repress the expression of a group of target genes. Using purified proteins, we demonstrate that a1 and alpha 2 form a highly specific DNA-binding activity, which recognizes an operator found upstream of each target gene. These proteins interact with DNA to form a ternary complex in which both a1 and alpha 2 contact the DNA through their respective homeodomains. An alpha 2 homodimer can recognize the same operator as the a1/alpha 2 heterodimer, but the affinity and specificity of the alpha 2 homodimer for DNA are much weaker than those of the a1/alpha 2 heterodimer. This difference results in part from the fact that the heterodimer is formed using a set of protein-protein contacts that is distinct from those that form the alpha 2/alpha 2 homodimer. Although a1 contacts DNA in the presence of alpha 2, it does not on its own bind DNA specifically, even at very high concentrations. These results suggest that the dimerization of heterologous partners can produce a DNA-binding activity that is not a simple hybrid of the known properties of each constituent.

Base Sequence↗