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Results for “Operator Regions, Genetic”

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At least 19 recordsLinked to original sources

PubMed2000

Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop.

The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter. MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point. In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and -10 region. Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression. Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter. The two MelR binding sites required for this optimal repression are separated by 177 bp. We suggest that, in the absence of melibiose, MelR forms a loop between these two sites. We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated. However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and -10 region. Parallels with the Escherichia coli araC-araBAD regulatory region are discussed.

Binding Sites↗

PubMed1996

Regulation of gene expression at a distance: the hypothetical role of regulatory protein-mediated topological changes of DNA.

A theoretical model is presented that a regulatory protein may activate the transcription of a promoter by interacting with a single remote operator. In response to an inducer molecule the regulatory protein bound to the operator undergoes a conformational change, and might mediate a B to Z-DNA conversion of the operator. This transition would remove both helical turns and supercoils from the intervening region between the operator and the promoter, resulting in the correct spatial arrangement of the -10 and -35 hexamers of the promoter, which therefore can be efficiently transcribed.

Agrobacterium tumefaciens↗

PubMed1993

A linguistic representation of the regulation of transcription initiation. I. An ordered array of complex symbols with distinctive features.

The inadequacy of context-free grammars in the description of regulatory information contained in DNA gave the formal justification for a linguistic approach to the study of gene regulation. Based on that result, we have initiated a linguistic formalization of the regulatory arrays of 107 sigma 70 E. coli promoters. The complete sequences of promoter (Pr), operator (Op) and activator binding sites (I) have previously been identified as the smallest elements, or categories, for a combinatorial analysis of the range of transcription initiation of sigma 70 promoters. These categories are conceptually equivalent to phonemes of natural language. Several features associated with these categories are required in a complete description of regulatory arrays of promoters. We have to select the best way to describe the properties that are pertinent for the description of such regulatory regions. In this paper we define distinctive features of regulatory regions based on the following criteria: identification of subclasses of substitutable elements, simplicity, selection of the most directly related information, and distinction of one array among the whole set of promoters. Alternative ways to represent distances in between regulatory sites are discussed, permitting, together with a principle of precedence, the identification of an ordered set of complex symbols as a unique representation for a promoter and its associated regulatory sites. In the accompanying paper additional distinctive features of promoters and regulatory sites are identified.

Binding Sites↗

PubMed1998

Evidence for two modes of cooperative DNA binding in vivo that do not involve direct protein-protein interactions.

BACKGROUND: The promoter regions of most eukaryotic genes contain binding sites for more than one transcriptional activator and these activators often bind cooperatively to promoters. The most common type of cooperativity is supported by direct protein-protein interactions. Recent studies have shown that proteins that do not specifically interact with one another can bind cooperatively to chromatin in vitro. probably by the localized destabilization of nucleosome structure by one factor, facilitating binding of another to a nearby site. This mechanism does not require that the transcription factors have activation domains. We have examined whether this phenomenon occurs in vivo. RESULTS: Unrelated non-interacting proteins can bind DNA cooperatively in yeast cells; this cooperative binding can contribute significantly to transcriptional activation, does not require that both factors have activation domains and is only operative over relatively short distances. In addition to this 'short-range' mechanism, unrelated non-interacting proteins can bind cooperatively to sites separated by hundreds of base pairs, so long as both have potent activation domains. CONCLUSION: Cooperative binding of transcription factors in vivo can occur by several mechanisms, some of which do not require direct protein-protein interactions and which cannot be detected in vitro using naked DNA templates. These findings must be taken into account when evaluating mechanisms for synergistic transcriptional activation.

Bacterial Proteins↗

PubMed1993

The elements for a classification of units of genetic information with a combinatorial component.

An integrative approach to the study of the regulation of gene expression has been undertaken here. The main goal of this approach is to make explicit the common rules that govern the relative location of regulatory sites within operons and other units of genetic information (UGIs). A classification that emphasizes the regulatory properties of UGIs can be achieved by partitioning UGIs into short sequences with defined properties. Such a classification scheme can be precisely defined as a Grammar with a component of combinatorial (rewriting) rules, and a dictionary component. Sequences have then to be grouped into classes such that any sequence of the same class can mutually substitute and produce novel regulatable UGIs. It is shown here that individual nucleotides cannot define such classes--they are far from equivalent to phonemes. Neither pairs, triplets or any short sequence with a defined number of nucleotides can define productive substitutions. Defined sequences like promoter, operator and activator binding sites are the smallest elements of combinatorial rules within the defined range of transcription initiation of sigma 70 Escherichia coli promoters.

Animals↗

PubMed2003

The role of dimerization in noise reduction of simple genetic networks.

Fluctuations are an intrinsic property of genetic networks due to the small number of interacting molecules. We study the role of dimerization reactions in controlling these fluctuations in a simple genetic circuit with negative feedback. We compare two different pathways. In the dimeric pathway the proteins to be regulated form dimers in solution that afterward bind to an operator site and inhibit transcription. In the monomeric pathway monomers bind to the operator site and then recruit another monomer to form a dimer directly on the DNA. We find that while both pathways implement the same negative feedback mechanism, the protein number fluctuations in the dimeric pathway are drastically reduced compared to the monomeric pathway. This difference in the ability to reduce fluctuations may be of importance in the design of genetic networks.

Algorithms↗

PubMed1987

Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters.

We present a statistical-mechanical selection theory for the sequence analysis of a set of specific DNA regulatory sites that makes it possible to predict the relationship between individual base-pair choices in the site and specific activity (affinity). The theory is based on the assumption that specific DNA sequences have been selected to conform to some requirement for protein binding (or activity), and that all sequences that can fulfil this requirement are equally likely to occur. In most cases, the number of specific DNA sequences that are known for a certain DNA-binding protein is very small, and we discuss in detail the small-sample uncertainties that this leads to. When applied to the binding sites for cro repressor in phage lambda, the theory can predict, from the sequence statistics alone, their rank order binding affinities in reasonable agreement with measured values. However, the statistical uncertainty generated by such a small sample (only 6 sites known) limits the result to order-of-magnitude comparisons. When applied to the much larger sample of Escherichia coli promoter sequences, the theory predicts the correlation between in vitro activity (k2KB values) and homology score (closeness to the consensus sequence) observed by Mulligan et al. (1984). The analysis of base-pair frequencies in the promoter sample is consistent with the assumption that base-pairs at different positions in the sites contribute independently to the specific activity, except in a few marginal cases that are discussed. When the promoter sites are ordered according to predicted activities, they seem to conform to the Gaussian distribution that results from a requirement for maximal sequence variability within the constraint of providing a certain average activity. The theory allows us to compare the number of specific sites with a certain activity to the number that would be expected from random occurrence in the genome. While strong promoters are "overspecified", in the sense that their probability of random occurrence is very low, random sequences with weak promoter-like properties are expected to occur in very large numbers. This leads to the conclusion that functional specificity is based on other properties in addition to primary sequence recognition; some possibilities are discussed. Finally, we show that the sequence information, as defined by Schneider et al. (1986), can be used directly (at least in the case of equilibrium binding sites) to estimate the number of protein molecules that are specifically bound at random "pseudosites" in the genome.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗

PubMed1989

Lambda cro repressor complex with OR3 operator DNA. 19F nuclear magnetic resonance observations.

The interaction of lambda cro repressor with DNA is probed using synthetic 17 base-pair OR3 operators in which 5-fluorodeoxyuridine has been systematically incorporated at each of the nine positions normally occupied by a thymidine residue. By monitoring changes in chemical shift of the fluorine resonances upon cro repressor binding in aqueous buffers of varying 2H2O content, we have examined the specific cro repressor-OR3 DNA complex in detail. The results are interpreted in the context of the popular model for cro repressor-OR3 complex derived from the three-dimensional structure of the cro repressor in the absence of DNA. The results presented here not originally predicted by the model are: (1) there is an asymmetry in the environment at the two ends of the operator, although the base-pairs involved and the cro repressor dimer are symmetric; (2) there appears to be distortion of the DNA helix at two distinct positions; (3) changes of the DNA environment in the middle of the helix suggest additional DNA distortion not near the contact areas proposed in the model.

Bacteriophage lambda↗

PubMed1993

A linguistic representation of the regulation of transcription initiation. II. Distinctive features of sigma 70 promoters and their regulatory binding sites.

The goal of this paper and the accompanying one is to achieve a linguistic representation of a set of sigma 70 promoters. Such a description is formed by an ordered concatenated array of complex symbols identified by their categorical property, i.e. promoter, operator, activator binding site, etc. Each of these symbols may contain several properties associated with their respective classes of 'molecular words'. The main problem in attaining such a description is to define which properties are going to be represented, and how. In the accompanying paper the criteria on which the selection of alternative descriptions is based were discussed. The properties of promoters and regulatory sites are discussed here, and their corresponding distinctive features are selected following such criteria. Thus, information that is not directly relevant and that can overspecify the description has been excluded, since it does not seem to contribute to identifying classes of substitutable elements. Other properties, such as strength of promoters, position of regulatory sites, different types of specificities of regulatory proteins, affinity of their binding sites, etc., are also discussed. As a result of this analysis, a complete representation with distinctive features of the set of sigma 70 promoters is attainable.

Binding Sites↗

PubMed2005

Real-time kinetics of gene activity in individual bacteria.

Protein levels have been shown to vary substantially between individual cells in clonal populations. In prokaryotes, the contribution to such fluctuations from the inherent randomness of gene expression has largely been attributed to having just a few transcripts of the corresponding mRNAs. By contrast, eukaryotic studies tend to emphasize chromatin remodeling and burst-like transcription. Here, we study single-cell transcription in Escherichia coli by measuring mRNA levels in individual living cells. The results directly demonstrate transcriptional bursting, similar to that indirectly inferred for eukaryotes. We also measure mRNA partitioning at cell division and correlate mRNA and protein levels in single cells. Partitioning is approximately binomial, and mRNA-protein correlations are weaker earlier in the cell cycle, where cell division has recently randomized the relative concentrations. Our methods further extend protein-based approaches by counting the integer-valued number of transcript with single-molecule resolution. This greatly facilitates kinetic interpretations in terms of the integer-valued random processes that produce the fluctuations.

Algorithms↗

PubMed2006

Lambda's switch: lessons from a module swap.

A recent experiment has replaced Cro, a crucial component of lambda's genetic switch, with the lac repressor (plus two lac operators). The resulting hybrid phage is viable, but a subtle phenotypic defect explains a puzzle concerning the workings of the switch.

Bacteriophage lambda↗

PubMed2005

Logic functions of the genomic cis-regulatory code.

cis-Regulatory modules that control developmental gene expression process the regulatory inputs provided by the transcription factors for which they contain specific target sites. A prominent class of cis-regulatory processing functions can be modeled as logic operations. Many of these are combinatorial because they are mediated by multiple sites, although others are unitary. In this work, we illustrate the repertoire of cis-regulatory logic operations, as an approach toward a functional interpretation of the genomic regulatory code.

Animals↗

PubMed1989

Phage lambda Cro protein and cI repressor use two different patterns of specific protein-DNA interactions to achieve sequence specificity in vivo.

By assaying the binding of wild-type Cro to a set of 40 mutant lambda operators in vivo, we have determined that the 14 outermost base pairs of the 17 base pair, consensus lambda operator are critical for Cro binding. Cro protein recognizes 4 base pairs in a lambda operator half-site in different ways than cI repressor. The sequence determinants of Cro binding at these critical positions in vivo are nearly perfectly consistent with the model proposed by W. F. ANDERSON, D. H. OHLENDORF, Y. TAKEDA and B. W. MATTHEWS and modified by Y. TAKEDA, A. SARAI and V. M. RIVERA for the specific interactions between Cro and its operator, and explain the relative order of affinities of the six natural lambda operators for Cro. Our data call into question the idea that lambda repressor and Cro protein recognize the consensus lambda operator by nearly identical patterns of specific interactions.

Amino Acids↗

PubMed2004

Quantitative modeling of DNA-protein interactions: effects of amino acid substitutions on binding specificity of the Mnt repressor.

Understanding DNA-protein recognition quantitatively is essential to developing computational algorithms for accurate transcriptional binding site prediction. Using a quantitative, multiple fluorescence, relative affinity (QuMFRA) assay, we determine the binding specificity of 11 different position 6 variants of the Mnt repressor for operators containing all 16 possible dinucleotides at operator positions 16 and 17. We show that the wild-type and all variant proteins interact with the two positions in a non-independent manner, but that a simple independent model provides a close approximation to the true binding affinities. The wild-type His at amino acid 6 is the only protein to prefer the AC sequence of the wild-type operator, whereas most of the variant proteins prefer TA. H6R is unique in having a strong preference for C at position 16. A comparison of the quantitative binding data for all of the protein variants with a model for recognition of the early growth response (EGR) zinc finger family suggests that interactions of Mnt with positions 16 and 17 are similar to interactions of EGR with positions 1 and 2, respectively. This information leads to an augmented model for the interaction of Mnt with its operator.

Amino Acid Substitution↗

PubMed1989

Interaction of distinct domains in Mu transposase with Mu DNA ends and an internal transpositional enhancer.

Bacteriophage Mu is the largest and most efficient transposable element known. The Mu transposase (A protein) of relative molecular mass 75,000 is a central component of the transposition machinery. We report here that the N-terminal region of Mu transposase contains two distinct DNA-binding domains, one which binds the two Mu DNA ends, and another which binds an internal operator region. This internal operator is required for the transposase-mediated synapsis and nicking of Mu ends in vitro, and stimulates transposition more than 100-fold in vivo. The orientation of the operator with respect to the ends is critical to its function, whereas its distance from the ends seems to be relatively unimportant. We propose that the operator enhances transposition by transiently interacting with the transposase and Mu DNA end(s) to form a complex in which synapsis of the ends occurs.

Bacteriophages↗

PubMed2000

Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding II: cooperative interactions of cro dimers.

The bacteriophage lambda relies on interactions of the cI and cro repressors which self assemble and bind the two operators (O(R) and O(L)) of the phage genome to control the lysogenic to lytic switch. While the self assembly and O(R) binding of cI have been investigated in detail, a more complete understanding of gene regulation by phage lambda also requires detailed knowledge of the role of cro repressor as it dimerizes and binds at O(R) sites. Since dimerization and operator binding are coupled processes, a full elucidation of the regulatory energetics in this system requires that the equilibrium constants for dimerization and cooperative binding be determined. The dimerization constant for cro has been measured as a prelude to these binding studies. Here, the energetics of cro binding to O(R) are evaluated using quantitative DNaseI footprint titration techniques. Binding data for wild-type and modified O(R) site combinations have been simultaneously analyzed in concert with the dimerization energetics to obtain both the intrinsic and cooperative DNA binding energies for cro with the three O(R) sites. Binding of cro dimers is strongest to O(R)3, then O(R)1 and lastly, O(R)2. Adjacently bound repressors exhibit positive cooperativity ranging from -0.6 to -1.0 kcal/mol. Implications of these, newly resolved, energetics are discussed in the framework of a dynamic model for gene regulation. This characterization of the DNA-binding properties of cro repressor establishes the foundation on which the system can be explored for other, more complex, regulatory elements such as cI-cro cooperativity.

Allosteric Site↗

PubMed2002

Thermodynamics of E. coli cytidine repressor interactions with DNA: distinct modes of binding to different operators suggests a role in differential gene regulation.

Interactions between the Escherichia coli cytidine repressor protein (CytR) and its operator sites at the different promoters that comprise the CytR regulon, play an important role in the regulation of these promoters. The natural operators are palindromes separated by variable length central spacers (0-9 bp). We have suggested that this variability affects the flexibility of CytR-DNA contacts, thereby affecting the critical protein-protein interactions between CytR and the cAMP receptor protein (CRP) that underlie differential repression and activation of CytR-regulated genes. To assess this hypothesis, we investigated the thermodynamics of CytR binding to the natural operator sequences found in udpP and deoP2. To separate effects due to spacing from effects due to the differing sequences of the recognition half-sites of these two operators, we also investigated CytR binding to artificial hybrid operators, in which the half-site sequences of udpP and deoP2 were exchanged. Thermodynamic parameters, DeltaS(o), DeltaH(o) and DeltaC(o)(p), were determined by van't Hoff analysis of CytR binding, monitored by changes in the steady-state fluorescence anisotropy of dye-conjugated, operator-containing oligonucleotides. Large differences in thermodynamics were observed that depend primarily on the central spacer rather than the sequences of the recognition half-sites. Binding to operators with deoP2 spacing results in a very large, negative DeltaC(o)(p). Association is strongly favored enthalpically and strongly disfavored entropically at ambient temperature. By contrast, binding to operators with udpP spacing results in a small, negative DeltaC(o)(p). Association is weakly favored both enthalpically and entropically at ambient temperature. A difference of such magnitude in DeltaDeltaC(o)(p) has not been reported previously for specific binding of a transcription factor to different sites. The identical salt dependence of CytR binding to deoP2 and udpP operators indicates that ion-dependent processes do not contribute significantly to this difference. Thus, the different thermodynamic effects appear to reflect distinctly different modes of site-specific DNA binding. We discuss similarities to operator binding by CytR homologs among LacI family repressors, and we consider how different CytR binding modes might affect interactions with other components of the gene regulatory machinery that contribute to differential gene regulation.

Base Sequence↗

PubMed1997

Regulation by tetracycline of gene expression in Saccharomyces cerevisiae.

A convenient system for the control of gene expression in Saccharomyces cerevisiae was developed. Tetracycline-responsive promoters were constructed by fusing the tetracycline operator (tetO) to the S. cerevisiae HOP1 promoter. When fused to the tetracycline repressor (tetR), trans-activation domains of both GAL4 and HAP4 were capable of promoting transcription from the tetO-HOP1 chimeric promoter, but the tetR-HAP4 fusion activator was the more efficient transcriptional activator. Addition of tetracycline nearly completely repressed activator-dependent transcription from the tetO-HOP1 promoter. Moreover, tetracycline-dependent repression of YEF3, CDC28 and RAM2 expression impaired cell growth. Thus, this system is useful for the elucidation of gene function in S. cerevisiae.

Gene Expression Regulation, Fungal↗