ORCID Profile
0000-0003-2969-6841
Current Organisation
University of Adelaide
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Publisher: American Physical Society (APS)
Date: 08-12-2006
Publisher: Public Library of Science (PLoS)
Date: 11-07-2008
Publisher: Cold Spring Harbor Laboratory
Date: 27-09-2019
DOI: 10.1101/784983
Abstract: The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator binding to the phage attachment site ( att ), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA, and spreads from specific binding sites into adjacent non-specific DNA features that are shared with other RDFs. By analysing Apl’s repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.
Publisher: Wiley
Date: 25-07-2002
DOI: 10.1046/J.1365-2958.2002.03038.X
Abstract: The non-lambdoid coliphage 186 provides an alternative model to the lytic-lysogenic switch of phage lambda. Like lambda, the key switch regulator, the CI repressor, associates to octamers. Unlike lambda, the lytic promoter (pR) and the lysogenic promoter (pL) are face-to-face, 62 bp apart and are flanked by distal CI binding sites (FL and FR) located approximately 300 bp away. Using reporter and footprinting studies, we show that the outcome, but not the mechanism, of regulation by 186 CI is very similar to lambda. 186 CI stimulates pL transcription indirectly by repressing convergent interfering transcription from pR. However, in the absence of the flanking FL and FR sites, CI bound at pR interacts co-operatively with a weak CI binding site at pL and represses both promoters. FL and FR play a critical role they assist repression of pR and simultaneously alleviate repression of pL, thus allowing high pL activity. We propose that the 186 switch is regulated by a novel mechanism in which a CI octamer bound at pR forms alternative DNA loops to pL or to a flanking site, depending on CI concentration.
Publisher: Cold Spring Harbor Laboratory
Date: 10-2007
DOI: 10.1101/GAD.1584907
Abstract: CI represses cro Cro represses cI . This double negative feedback loop is the core of the classical CI–Cro epigenetic switch of bacteriophage λ. Despite the classical status of this switch, the role in λ development of Cro repression of the P RM promoter for CI has remained unclear. To address this, we created binding site mutations that strongly impaired Cro repression of P RM with only minimal effects on CI regulation of P RM . These mutations had little impact on λ development after infection but strongly inhibited the transition from lysogeny to the lytic pathway. We demonstrate that following inactivation of CI by ultraviolet treatment of lysogens, repression of P RM by Cro is needed to prevent synthesis of new CI that would otherwise significantly impede lytic development. Thus a bistable CI–Cro circuit reinforces the commitment to a developmental transition.
Publisher: Elsevier BV
Date: 11-2014
Publisher: Elsevier BV
Date: 12-2011
DOI: 10.1016/J.JMB.2011.10.027
Abstract: Positive feedback in nucleosome modification has been proposed to allow large chromatin regions to exist stably and heritably in distinct expression states. However, modeling has shown that such epigenetic bistability requires that modifying enzymes recruited by nucleosomes are active on distant nucleosomes, potentially allowing uncontrollable spreading of modification. By modeling the silencing of mating-type loci in Saccharomyces cerevisiae, we show that a modification reaction that combines a long-range component and a locally acting component can provide bistability and can be blocked by simple barriers that interrupt the nucleosome chain. We find that robust containment of the silenced region could be achieved by the presence of a number of weak simple barriers in the surrounding chromatin and a limited capacity of the positive feedback reaction. In addition, we show that the state of the silenced region can be regulated by silencer elements acting only on neighboring nucleosomes. Thus, a relatively simple set of nucleosome-modifying enzymes and recognition domains is all that is needed to make chromatin-based epigenetics useful and safe.
Publisher: Cold Spring Harbor Laboratory
Date: 15-11-2001
DOI: 10.1101/GAD.937301
Abstract: The CI repressor of bacteriophage λ is a model for the role of cooperativity in the efficient functioning of genetic switches. Pairs of CI dimers interact to cooperatively occupy adjacent operator sites at O R and at O L . These CI tetramers repress the lytic promoters and activate transcription of the cI gene from P RM . CI is also able to octamerize, forming a large DNA loop between O R and O L , but the physiological role of this is unclear. Another puzzle is that, although a dimer of CI is able to repress P RM by binding to the third operator at O R , O R 3, this binding seems too weak to affect CI production in the lysogenic state. Here we show that repression of P RM at lysogenic CI concentrations is absolutely dependent on O L , in this case 3.8 kb away. A mutant defective in this CI negative autoregulation forms a lysogen with elevated CI levels that cannot efficiently switch from lysogeny to lytic development. Our results invalidate previous evidence that Cro binding to O R 3 is important in prophage induction. We propose the octameric CI: O R – O L complex increases the affinity of CI for O R 3 by allowing a CI tetramer to link O R 3 and the third operator at O L , O L 3.
Publisher: Oxford University Press (OUP)
Date: 1988
Abstract: The importance of the preschool period for becoming a skilled reader is highlighted by a significant body of evidence that preschool children's development in the areas of oral language, phonological awareness, and print knowledge is predictive of how well they will learn to read once they are exposed to formal reading instruction in elementary school. Although there are now a number of empirically supported instructional activities for helping children who are at -risk of later reading difficulties acquire these early literacy skills, limitations in instructional time and opportunities in most preschool settings requires the use of valid assessment procedures to ensure that instructional resources are utilized efficiently. In this paper, we discuss the degree to which informal, diagnostic, screening, and progress-monitoring assessments of preschool early literacy skills can inform instructional decisions by considering the strengths and weaknesses of each approach to assessment.
Publisher: American Society for Microbiology
Date: 29-10-2010
Abstract: Genes of prokaryotes and Archaea are often organized in cotranscribed groups, or operons. In contrast, eukaryotic genes are generally transcribed independently. Here we show that there is a substantial economic gain for the cell to cotranscribe genes encoding protein complexes because it synchronizes the fluctuations, or noise, in the levels of the different components. This correlation substantially reduces the shortfall in production of the complex. This benefit is relatively large in small cells such as bacterial cells, in which there are few mRNAs and proteins per cell, and is diminished in larger cells such as eukaryotic cells.
Publisher: Elsevier BV
Date: 09-1986
DOI: 10.1016/0022-2836(86)90257-3
Abstract: The PstI fragment (65.5% to 77.4%) of coliphage 186, known genetically to encode the major control genes, has been sequenced, and an analysis performed to assess coding capacity, transcription-translation signals, and to identify any other significant features. Our analysis indicates that the region encodes: seven genes, including the int and cI genes, which overlap, the late control gene B, and two genes, named CP75 and CP76, encoding potential DNA-binding proteins a promoter pB and terminator tB for the rightward transcription of the B gene, and we predict the existence of this transcript in a lysogen a promoter pL and terminator tL for leftward transcription that encodes the int and cI genes, and represents the presumed lysogenic transcript a promoter pR for rightward transcription to give the presumed (early) lytic transcript that is overlapping and convergent with the lysogenic transcript and finally, a potential operator site for repressor binding in the region of the pR promoter. Preliminary evidence is presented to support this analysis.
Publisher: Elsevier BV
Date: 04-2005
DOI: 10.1016/J.GDE.2005.02.001
Abstract: The contribution of bacteriophage lambda to gene control research is far from over. A revised model of the lambda genetic switch includes extra cooperativity through octamerization of the cI repressor protein, mediated by long-range DNA looping. Structural analysis reveals remarkably subtle transcriptional activation by cI. The action of cI, activation by cII, and aspects of antitermination by N and Q all confirm the utility and versatility of simple, weak adhesive interactions mediated by nucleic acid tethers. New genetic and quantitative analysis of the lambda gene network is challenging cherished ideas about how complex behaviours emerge from this regulatory system.
Publisher: Cold Spring Harbor Laboratory
Date: 02-2004
DOI: 10.1101/GAD.1167904
Abstract: Effective repression of cI transcription from P RM by the bacteriophage λ CI repressor requires binding sites ( O L ) located 2.4 kb from the promoter. A CI tetramer bound to O L 1 .O L 2 interacts with a tetramer bound near P RM ( O R 1 .O R 2), looping the intervening DNA. We previously proposed that in this CI octamer:DNA complex, the distant O L 3 operator and the weak O R 3 operator overlapping P RM are juxtaposed so that a CI dimer at O L 3 can cooperate with a CI dimer binding to O R 3. Here we show that O L 3 is necessary for effective repression of P RM and that the repressor at O L 3 appears to interact specifically with the repressor at O R 3. The O L 3-CI- O R 3 interaction involves the same CI interface used for short-range dimer-dimer interactions and does not occur without the other four operators. The long-range interactions were incorporated into a physicochemical model, allowing estimation of the long-range interaction energies and showing the lysogenic state to be ideally poised for CI negative autoregulation. The results establish the λ system as a powerful tool for examining long-range gene regulatory interactions in vivo.
Publisher: Oxford University Press (OUP)
Date: 04-02-2017
DOI: 10.1093/NAR/GKX069
Publisher: Elsevier BV
Date: 05-1996
Publisher: Oxford University Press (OUP)
Date: 17-01-2011
DOI: 10.1093/NAR/GKQ1336
Publisher: Oxford University Press (OUP)
Date: 19-11-2021
DOI: 10.1093/NAR/GKAA1065
Abstract: The CII protein of temperate coliphage 186, like the unrelated CII protein of phage λ, is a transcriptional activator that primes expression of the CI immunity repressor and is critical for efficient establishment of lysogeny. 186-CII is also highly unstable, and we show that in vivo degradation is mediated by both FtsH and RseP. We investigated the role of CII instability by constructing a 186 phage encoding a protease resistant CII. The stabilised-CII phage was defective in the lysis-lysogeny decision: choosing lysogeny with close to 100% frequency after infection, and forming prophages that were defective in entering lytic development after UV treatment. While lysogenic CI concentration was unaffected by CII stabilisation, lysogenic transcription and CI expression was elevated after UV. A stochastic model of the 186 network after infection indicated that an unstable CII allowed a rapid increase in CI expression without a large overshoot of the lysogenic level, suggesting that instability enables a decisive commitment to lysogeny with a rapid attainment of sensitivity to prophage induction.
Publisher: Elsevier BV
Date: 05-1996
Abstract: We have sequenced the LexA-controlled operon of coliphage 186 that carries the tum gene, whose product is necessary for UV induction of the 186 prophage. The operon consists of orf95 and orf97, and we have identified orf95 as the tum gene. The major translation products from orf95 result from internal initiations and modulate Tum activity. Tum is the product of the full-length Orf95 protein. The second gene of the operon, orf97, is of unknown function but, while it has little effect on prophage induction, its presence in the cell totally blocks infection of that cell by 186.
Publisher: Elsevier BV
Date: 05-1996
Abstract: Retrons are unusual, reverse transcriptase-encoding elements found in bacteria. Although there are a number of indications that retrons are mobile elements, their transposition has not been observed. The Escherichia coli retrons Ec67 and Ec86 are different retrons inserted at the same site and we have further characterized this site in search of clues to the mechanism of retron transposition. We confirm, by extending previous sequence analysis, that Ec67 and Ec86 are inserted into prophages related to coliphage 186. Comparison with the recently published sequence of the 186 96-2% region indicates that the retrons have replaced approximately 180 bp of DNA between the phage cohesive end site (cos) and the transcription terminator of a phage DNA-packaging gene. These features--DNA replacement at the insertion site and the location of retron junctions near transcription terminators or DNA cleavage sites--are shared with other retrons and suggest ways in which retron transposition might have occurred.
Publisher: Wiley
Date: 29-03-2019
Publisher: Informa UK Limited
Date: 09-2013
DOI: 10.4161/TRNS.26101
Publisher: EMBO
Date: 2008
DOI: 10.1038/MSB.2008.21
Publisher: Springer Science and Business Media LLC
Date: 20-11-2017
DOI: 10.1038/S41467-017-01873-X
Abstract: DNA looping is a ubiquitous and critical feature of gene regulation. Although DNA looping can be efficiently detected, tools to readily manipulate DNA looping are limited. Here we develop CRISPR-based DNA looping reagents for creation of programmable DNA loops. Cleavage-defective Cas9 proteins of different specificity are linked by heterodimerization or translational fusion to create bivalent complexes able to link two separate DNA regions. After model-directed optimization, the reagents are validated using a quantitative DNA looping assay in E. coli . Looping efficiency is ~15% for a 4.7 kb loop, but is significantly improved by loop multiplexing with additional guides. Bivalent dCas9 complexes are also used to activate endogenous norVW genes by rewiring chromosomal DNA to bring distal enhancer elements to the gene promoters. Such reagents should allow manipulation of DNA looping in a variety of cell types, aiding understanding of endogenous loops and enabling creation of new regulatory connections.
Publisher: Elsevier BV
Date: 02-2002
Publisher: Oxford University Press (OUP)
Date: 27-11-2013
DOI: 10.1093/NAR/GKT1235
Abstract: Inheritance of 5-methyl cytosine modification of CpG (CG/CG) DNA sequences is needed to maintain early developmental decisions in vertebrates. The standard inheritance model treats CpGs as independent, with methylated CpGs maintained by efficient methylation of hemimethylated CpGs produced after DNA replication, and unmethylated CpGs maintained by an absence of de novo methylation. By stochastic simulations of CpG islands over multiple cell cycles and systematic s ling of reaction parameters, we show that the standard model is inconsistent with many experimental observations. In contrast, dynamic collaboration between CpGs can provide strong error-tolerant somatic inheritance of both hypermethylated and hypomethylated states of a cluster of CpGs, reproducing observed stable bimodal methylation patterns. Known recruitment of methylating enzymes by methylated CpGs could provide the necessary collaboration, but we predict that recruitment of demethylating enzymes by unmethylated CpGs strengthens inheritance and allows CpG islands to remain hypomethylated within a sea of hypermethylation.
Publisher: Oxford University Press (OUP)
Date: 17-07-2014
DOI: 10.1093/NAR/GKU627
Publisher: Public Library of Science (PLoS)
Date: 20-12-2010
Publisher: Public Library of Science (PLoS)
Date: 16-08-2012
Publisher: American Society for Microbiology
Date: 03-2012
DOI: 10.1128/JVI.07236-11
Publisher: Public Library of Science (PLoS)
Date: 23-12-2005
Publisher: Springer Science and Business Media LLC
Date: 06-2023
DOI: 10.1007/S12551-023-01064-7
Abstract: In recent years, transcriptional roadblocking has emerged as a crucial regulatory mechanism in gene expression, whereby other DNA-bound obstacles can block the progression of transcribing RNA polymerase (RNAP), leading to RNAP pausing and ultimately dissociation from the DNA template. In this review, we discuss the mechanisms by which transcriptional roadblocks can impede RNAP progression, as well as how RNAP can overcome these obstacles to continue transcription. We examine different DNA-binding proteins involved in transcriptional roadblocking and their biophysical properties that determine their effectiveness in blocking RNAP progression. The catalytically dead CRISPR-Cas (dCas) protein is used as an ex le of an engineered programmable roadblock, and the current literature in understanding the polarity of dCas roadblocking is also discussed. Finally, we delve into a stochastic model of transcriptional roadblocking and highlight the importance of transcription factor binding kinetics and its resistance to dislodgement by an elongating RNAP in determining the strength of a roadblock.
Publisher: Oxford University Press (OUP)
Date: 29-02-2016
DOI: 10.1093/NAR/GKW124
Publisher: Elsevier BV
Date: 03-2000
DOI: 10.1016/J.JMB.2004.11.075
Abstract: Interactions between RNA polymerases (RNAP) resulting from tandem or convergent arrangements of promoters can cause transcriptional interference, often with important consequences for gene expression. However, it is not known what factors determine the magnitude of interference and which mechanisms are likely to predominate in any situation. We therefore developed a mathematical model incorporating three mechanisms of transcriptional interference in bacteria: occlusion (in which passing RNAPs block access to the promoter), collisions between elongating RNAPs, and "sitting duck" interference (in which RNAP complexes waiting to fire at the promoter are removed by passing RNAP). The predictions of the model are in good agreement with a recent quantitative in vivo study of convergent promoters in E.coli. Our analysis predicts that strong occlusion requires the interfering promoter to be very strong. Collisions can also produce strong interference but only if the interfering promoter is very strong or if the convergent promoters are far apart (>200 bp). For moderate strength interfering promoters and short inter-promoter distances, strong interference is dependent on the sitting duck mechanism. Sitting duck interference is dependent on the relative strengths of the two promoters. However, it is also dependent on the "aspect ratio" (the relative rates of RNAP binding and firing) of the sensitive promoter, allowing promoters of equal strength to have very different sensitivities to transcriptional interference. The model provides a framework for using transcriptional interference to investigate various dynamic processes on DNA in vivo.
Publisher: Elsevier BV
Date: 02-2019
DOI: 10.1016/J.CELREP.2019.02.002
Abstract: Enhancers are ubiquitous and critical gene-regulatory elements. However, quantitative understanding of the role of DNA looping in the regulation of enhancer action and specificity is limited. We used the Escherichia coli NtrC enhancer-σ54 promoter system as an in vivo model, finding that NtrC activation is highly sensitive to the enhancer-promoter (E-P) distance in the 300-6,000 bp range. DNA loops formed by Lac repressor were able to strongly regulate enhancer action either positively or negatively, recapitulating promoter targeting and insulation. A single LacI loop combining targeting and insulation produced a strong shift in specificity for enhancer choice between two σ54 promoters. A combined kinetic-thermodynamic model was used to quantify the effect of DNA-looping interactions on promoter activity and revealed that sensitivity to E-P distance and to control by other loops is itself dependent on enhancer and promoter parameters that may be subject to regulation.
Publisher: Elsevier BV
Date: 03-2006
DOI: 10.1016/J.MOLCEL.2006.01.019
Abstract: Bacteriophage lambda is a paradigm for understanding the role of cooperativity in gene regulation. Comparison of the regulatory regions of lambda and the unrelated temperate bacteriophage 186 provides insight into alternate ways to assemble functional genetic switches. The structure of the C-terminal domain of the 186 repressor, determined at 2.7 A resolution, reveals an unusual heptamer of dimers, consistent with presented genetic studies. In addition, the structure of a cooperativity mutant of the full-length 186 repressor, identified by genetic screens, was solved to 1.95 A resolution. These structures provide a molecular basis for understanding lysogenic regulation in 186. Whereas the overall fold of the 186 and lambda repressor monomers is remarkably similar, the way the two repressors cooperatively assemble is quite different and explains in part the differences in their regulatory activity.
Publisher: Oxford University Press (OUP)
Date: 24-04-2013
DOI: 10.1093/NAR/GKT298
Publisher: Elsevier BV
Date: 08-1998
Abstract: The late-lytic region of the genome of bacteriophage 186 encodes the phage proteins that synthesize the complex viral particle and lyse the bacterial host. We report the completion of the DNA sequence of the late region and the assignment of 18 previously identified genes to open reading frames in the sequence. The 186 late region is similar to the late region of phage P2, sharing 26 genes of known function: the single gene for activation of late gene transcription, 6 genes for construction of DNA-containing heads, 16 for tail morphogenesis, and 3 for cell lysis. We identified two 186 late genes with unknown function one is homologous to previously unrecognised genes in P2, HP1, and phiCTX, and the other may modulate DNA packaging. The 186 late region, like the rest of the genome, lacks the lysogenic conversion genes that are carried by P2, allowing the 186 late region to be transcribed from only three late promoters rather than four. The relative absence of lysogenic conversion genes in 186 suggests that the two phages have evolved to use the lytic and lysogenic reproductive modes to different extents.
Publisher: Elsevier BV
Date: 06-2007
DOI: 10.1016/J.JMB.2007.04.041
Abstract: We describe a hybrid statistical mechanical and dynamical approach for modelling the formation of closed, open and elongating complexes of RNA polymerase, the interactions of these polymerases to produce transcriptional interference, and the regulation of these processes by a DNA-binding and DNA-looping regulatory protein. As a model system, we have used bacteriophage 186, for which genetic, biochemical and structural studies have suggested that the CI repressor binds as a 14-mer to form alternative DNA-looped complexes, and activates lysogenic transcription indirectly by relieving transcriptional interference caused by the convergent lytic promoter. The modelling showed that the original mechanisms proposed to explain this relief of transcriptional interference are not consistent with the available in vivo reporter data. However, a good fit to the reporter data was given by a revised model that incorporates a novel predicted regulatory mechanism: that RNA polymerase bound at the lysogenic promoter protects itself from transcriptional interference by recruiting CI to the lytic promoter. This mechanism and various estimates of in vivo biochemical parameters for the 186 CI system should be testable. Our results demonstrate the power of mathematical modelling for the extraction of detailed biochemical information from in vivo data.
Publisher: Oxford University Press (OUP)
Date: 04-07-2016
DOI: 10.1093/NAR/GKW600
Publisher: American Society for Microbiology
Date: 15-11-2009
DOI: 10.1128/JVI.01057-09
Abstract: Phage lambda is among the simplest organisms that make a developmental decision. An infected bacterium goes either into the lytic state, where the phage particles rapidly replicate and eventually lyse the cell, or into a lysogenic state, where the phage goes dormant and replicates along with the cell. Experimental observations by P. Kourilsky are consistent with a single phage infection deterministically choosing lysis and double infection resulting in a stochastic choice. We argue that the phage are playing a “game” of minimizing the chance of extinction and that the shift from determinism to stochasticity is due to a shift from a single-player to a multiplayer game. Crucial to the argument is the clonal identity of the phage.
Publisher: Elsevier
Date: 2017
Publisher: Informa UK Limited
Date: 03-2017
Publisher: Proceedings of the National Academy of Sciences
Date: 04-02-2013
Abstract: How distant enhancer elements regulate the assembly of a transcription complex at a promoter remains poorly understood. Here, we use long-range gene regulation by the bacteriophage λ CI protein as a powerful system to examine this process in vivo. A 2.3-kb DNA loop, formed by CI bridging its binding sites at OR and OL , is known already to enhance repression at the lysogenic promoter PRM , located at OR . Here, we show that CI looping also activates PRM by allowing the C-terminal domain of the α subunit of the RNA polymerase bound at PRM to contact a DNA site adjacent to the distal CI sites at OL . Our results establish OL as a multifaceted enhancer element, able to activate transcription from long distances independently of orientation and position. We develop a physicochemical model of our in vivo data and use it to show that the observed activation is consistent with a simple recruitment mechanism, where the α–C-terminal domain to DNA contact need only provide ∼2.7 kcal/mol of additional binding energy for RNA polymerase. Structural modeling of this complete enhancer–promoter complex reveals how the contact is achieved and regulated, and suggests that distal enhancer elements, once appropriately positioned at the promoter, can function in essentially the same way as proximal promoter elements.
Publisher: American Chemical Society (ACS)
Date: 20-05-2013
DOI: 10.1021/SB400021J
Abstract: We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids.
Publisher: Oxford University Press (OUP)
Date: 1990
Abstract: We present an update of our method for systematic detection and evaluation of potential helix-turn-helix DNA-binding motifs in protein sequences [Dodd, I. and Egan, J. B. (1987) J. Mol. Biol. 194, 557-564]. The new method is considerably more powerful, detecting approximately 50% more likely helix-turn-helix sequences without an increase in false predictions. This improvement is due almost entirely to the use of a much larger reference set of 91 presumed helix-turn-helix sequences. The scoring matrix derived from this reference set has been calibrated against a large protein sequence database so that the score obtained by a sequence can be used to give a practical estimation of the probability that the sequence is a helix-turn-helix motif.
Publisher: Elsevier BV
Date: 04-2004
Publisher: Elsevier BV
Date: 12-2009
DOI: 10.1016/J.JMB.2009.09.053
Abstract: The behavior of living systems is dependent on large dynamical gene regulatory networks (GRNs). However, the functioning of even the smallest GRNs is difficult to predict. The bistable GRN of bacteriophage lambda is able to count to make a decision between lysis and lysogeny on the basis of the number of phages infecting the cell, even though replication of the phage genome eliminates this initial difference. By simulating the behavior of a large number of random transcriptional GRNs, we show that a surprising variety of GRNs can carry out this complex task, including simple CI-Cro-like mutual repression networks. Thus, our study extends the repertoire of simple GRNs. Counterintuitively, the major effect of the addition of CII-like regulation, generally thought to be needed for counting by lambda, was to improve the ability of the networks to complete a simulated prophage induction. Our study suggests that additional regulatory mechanisms to decouple Cro and CII levels may exist in lambda and that infection counting could be widespread among temperate bacteriophages, many of which contain CI-Cro-like circuits.
Publisher: Oxford University Press (OUP)
Date: 13-08-2020
DOI: 10.1093/NAR/GKAA655
Abstract: The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.
Publisher: IOP Publishing
Date: 04-06-2010
DOI: 10.1088/1478-3975/7/2/026010
Abstract: Cells can often choose among several stably heritable phenotypes. Ex les are the expressions of genes in eukaryotic cells where long chromosomal regions can adopt persistent and heritable silenced or active states that may be associated with positive feedback in dynamic modification of nucleosomes. We generalize this mechanism in terms of bistability associated with valleys in an epigenetic landscape. A transfer matrix method was used to rigorously follow the system through the disruptive process of cell ision. This combined treatment of noisy dynamics both between and during cell ision provides an efficient way to calculate the stability of alternative states in a broad range of epigenetic systems.
Publisher: Wiley
Date: 12-1993
DOI: 10.1111/J.1365-2958.1993.TB00983.X
Abstract: The Apl protein of the temperature coliphage 186 represses transcription of the immunity repressor gene and down-regulates lytic transcription. It is shown here that an apl- mutant is competent for lytic development and establishes lysogeny normally but is defective in excision of the prophage. The Apl protein binds between the lytic and lysogenic promoters and also near the phage attachment site, suggesting that its role in excision is direct. Apl thus appears to act as an excisionase as well as a repressor. The pattern of Apl-induced DNase I enhancements indicates that the DNA is bent by Apl. Potential Apl recognition sequences are identified these sequences are directly repeated several times across each binding region and are spaced 10 or 11 bases apart, suggesting that Apl binds to one face of the DNA helix.
Publisher: American Physical Society (APS)
Date: 28-06-2016
Publisher: Oxford University Press (OUP)
Date: 28-01-2021
DOI: 10.1093/NAR/GKAA1284
Abstract: Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there are no general and technically simple assays to detect proteins capable of DNA looping in vivo nor to quantitate their in vivo looping efficiency. Here, we develop a quantitative in vivo assay for DNA-looping proteins in Escherichia coli that requires only basic DNA cloning techniques and a LacZ assay. The assay is based on loop assistance, where two binding sites for the candidate looping protein are inserted internally to a pair of operators for the E. coli LacI repressor. DNA looping between the sites shortens the effective distance between the lac operators, increasing LacI looping and strengthening its repression of a lacZ reporter gene. Analysis based on a general model for loop assistance enables quantitation of the strength of looping conferred by the protein and its binding sites. We use this ‘loopometer’ assay to measure DNA looping for a variety of bacterial and phage proteins.
Publisher: Proceedings of the National Academy of Sciences
Date: 06-10-2014
Abstract: Genes are frequently regulated by interactions between proteins that bind to the DNA near the gene and proteins that bind to DNA sites located far away, with the intervening DNA looped out. In eukaryotic genomes, genes and their distant sites are intermingled in complex ways and it is not understood how the correct connections are formed. Using two pairs of DNA-looping sites in bacterial cells, we tested the idea that one DNA loop can either assist or interfere with the formation of another DNA loop. By measuring the strength of these interactions between loops, we showed that this mechanism is capable of directing a distant site to the correct gene and preventing it contacting the wrong gene.
Publisher: Elsevier BV
Date: 04-1987
DOI: 10.1016/0022-2836(87)90681-4
Abstract: We have developed and tested a systematic method for the location and statistical evaluation of potential DNA-binding regions of the lambda Cro type in protein sequences. Using this approach to examine proteins expected to contain such regions, we have been able to compile a statistically homogeneous master set of 37 lambda Cro-like DNA-binding domains. Examination of a protein database revealed other prokaryotic proteins that are similar to this lambda Cro-like group. There are also many DNA-binding proteins that are not found to be significantly similar to the lambda Cro group, consistent with previous suggestions that different types of protein sequence may be able to achieve a similar mode of binding and that there exist other modes of sequence-specific DNA-binding. A useful feature of the method is that it can be applied without a computer.
Publisher: Proceedings of the National Academy of Sciences
Date: 16-12-2013
Abstract: Proteins bound to DNA often interact with proteins bound elsewhere on the same DNA to regulate gene expression. The intervening DNA tethers the proteins near each other, making their interaction efficient and specific, but the importance of this tethering effect is poorly understood at large DNA separations. We quantitated tethering inside bacterial cells, using two different proteins at separations up to 10,000 bp, to show that tethering is strong enough to drive efficient interactions over these distances. The same interactions were ∼10-fold weaker outside cells, implying that cellular factors enhance tethering. However, tethering was lost at a DNA separation of 500,000 bp inside bacteria, indicating special mechanisms inside eukaryotic cells to provide efficient and specific interactions over such distances.
Publisher: Informa UK Limited
Date: 04-2015
Publisher: Elsevier BV
Date: 07-1990
Publisher: Elsevier BV
Date: 06-2009
Publisher: Elsevier BV
Date: 05-2007
DOI: 10.1016/J.CELL.2007.02.053
Abstract: Chromosomal regions can adopt stable and heritable alternative states resulting in bistable gene expression without changes to the DNA sequence. Such epigenetic control is often associated with alternative covalent modifications of histones. The stability and heritability of the states are thought to involve positive feedback where modified nucleosomes recruit enzymes that similarly modify nearby nucleosomes. We developed a simplified stochastic model for dynamic nucleosome modification based on the silent mating-type region of the yeast Schizosaccharomyces pombe. We show that the mechanism can give strong bistability that is resistant both to high noise due to random gain or loss of nucleosome modifications and to random partitioning upon DNA replication. However, robust bistability required: (1) cooperativity, the activity of more than one modified nucleosome, in the modification reactions and (2) that nucleosomes occasionally stimulate modification beyond their neighbor nucleosomes, arguing against a simple continuous spreading of nucleosome modification.
No related grants have been discovered for Ian Dodd.