In Vivo Thermodynamic Analysis of Repression with and without Looping in lac Constructs

Law, Scott M.; Bellomy, Gregory R.; Schlax, Paula J.; Record, M. Thomas

doi:10.1006/jmbi.1993.1133

Cited by 87 publications

(65 citation statements)

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“…A helix repeat of 10.5 bp has also been found in DNA looping by bacteriophage cI and E. coli LacI repressors in vitro (6,7). Dependence of loop formation on the relative angular orientation of the two protein binding sites in a periodic fashion and thereby estimation of the size of the helix repeat has also been studied in several systems in vivo including an artificial construct in which a heterologous promoter was spanned by gal operators, O E and O I (4,8,10,29,30). The size of a DNA helical turn by in vivo estimation was shown to be within 11.0 -11.3 bp.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Repression of the gal Promoters by GalR and HU Depends on the Proper Helical Phasing of the Two Operators

Lewis

Adhya

2002

Journal of Biological Chemistry

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Repression of transcription initiation from the two gal promoters, P1 and P2, requires binding of GalR protein to two flanking operators, O E and O I , binding of HU to a site, hbs, located between the two operators, and supercoiled DNA template. Previous experiments suggested that repression involves the interaction of two DNA-bound GalR proteins, which generates a 113-bp DNA loop encompassing the promoter region. Interaction between two DNA-bound proteins would be allowed if the binding sites on DNA are properly aligned. To test the idea that the observed repression of gal transcription in vitro is mediated by DNA looping, we investigated the effect of changing the relative angular orientation of O E and O I in the DNA helix. We found that repression is a periodic function of the distance between the two operator sites. Since repression recurred commensurate with DNA helical repeat, we conclude that the observed in vitro repression is mediated by DNA looping and the in vitro conditions reflect the in vivo situation.DNA looping appears to be a general phenomenon in DNA transaction reactions e.g. replication, transcription regulation, site-specific recombination, DNA condensation, etc. In gene regulation, DNA looping is frequently involved in both transcription repression and activation. Looping occurs when two identical or different proteins bound to spatially separated specific sites on DNA interact directly or through a mediator or when a bidentate protein simultaneously binds to distal DNA sites. Depending on the strength of the above interactions, DNA looping may or may not require the aid of a DNA bending protein (frequently referred to as an architectural protein), which facilitates loop formation by binding to a locus between the two distal DNA sites. In the loop, the two binding sites on DNA may be oriented in parallel or in antiparallel orientation (1-3).The feasibility and stability of such a DNA-multiprotein complex has been studied in several systems, including the gal, lac, and ara operons and bacteriophage of Escherichia coli. The complex formation depends on a variety of conditions: the proper angular orientation of the protein binding sites on DNA (4 -8), the size of the loop (9), and the superhelicity of the DNA (10 -12). Because shorter DNA resists torsional change (13), the proper angular orientation of the two binding sites is more important with relatively shorter loop size than with longer loop size. It has been suggested that a loop size of less than 150 bp strictly depends upon proper phasing of the protein binding sites, less so for 200 bp, and not at all for 400 bp and up. The precise size of the helix repeat also depends to a certain extent on the DNA sequence of the loop (9). Here we report the results of the effect of changing the relative angular orientation of the binding sites, which were separated by less than 150 bp in DNA, on transcription repression in the gal operon of E. coli (14). Previous results suggested that repression involves the formation of a DNA loop by the intera...

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Section: Discussionmentioning

confidence: 99%

In Vitro Repression of the gal Promoters by GalR and HU Depends on the Proper Helical Phasing of the Two Operators

Lewis

Adhya

2002

Journal of Biological Chemistry

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“…The conceptual basis of this class of models is the idea that the expression level of the gene of interest can be deduced by examining the equilibrium probabilities that the DNA associated with that gene is occupied by various molecules -these include RNAP and a battery of transcription factors (TFs) such as repressors and activators. There is a long-standing tradition of using these ideas to unravel the dynamics of gene expression systems -particularly important examples being associated with the famed lac operon and phage lambda systems [18,[21][22][23][24][25][26]. Importantly, the thermodynamic models can serve as input to more general chemical kinetic models.…”

Section: How Much When and Where?mentioning

confidence: 99%

Transcriptional regulation by the numbers: models

Bintu

Buchler

García

et al. 2005

Current Opinion in Genetics & Development

707

884

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The expression of genes is regularly characterized with respect to how much, how fast, when and where. Such quantitative data demands quantitative models. Thermodynamic models are based on the assumption that the level of gene expression is proportional to the equilibrium probability that RNA polymerase (RNAP) is bound to the promoter of interest. Statistical mechanics provides a framework for computing these probabilities. Within this framework, interactions of activators, repressors, helper molecules and RNAP are described by a single function, the 'regulation factor'. This analysis culminates in an expression for the probability of RNA polymerase binding at the promoter of interest as a function of the number of regulatory proteins in the cell.

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“…We will focus on data in which the mechanical properties of DNA are used as an adjustable dial to tune a desired biological outcome during transcriptional regulation. In particular, we will address in vivo experiments like those performed by Müller et al, 101 Law et al, 102 and Becker et al, 103 where the level of repression is systematically measured as a function of the distance between two binding sites for Lac repressor ( Figure 7A inset). For reviews on Lac repressor refer to Matthews and Nichols 105 and to Lewis.…”

Section: Tightly Bent Dna In Transcriptional Regulationmentioning

confidence: 99%

“…Thermodynamic models of transcriptional regulation 111,112 have been used to extract the free energy of looping which is a measure of the cost of the looped configuration as a function of the distance between the operators. 102,[113][114][115] These models use equilibrium statistical mechanics to describe the probability of transcription as it is modulated by the presence of the repressor and its partner looped DNA.…”

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confidence: 99%

Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity

et al. 2006

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The mechanical properties of DNA play a critical role in many biological functions. For example, DNA packing in viruses involves confining the viral genome in a volume (the viral capsid) with dimensions that are comparable to the DNA persistence length. Similarly, eukaryotic DNA is packed in DNA-protein complexes (nucleosomes) in which DNA is tightly bent around protein spools. DNA is also tightly bent by many proteins that regulate transcription, resulting in a variation in gene expression that is amenable to quantitative analysis. In these cases, DNA loops are formed with lengths that are comparable to or smaller than the DNA persistence length. The aim of this review is to describe the physical forces associated with tightly bent DNA in all of these settings and to explore the biological consequences of such bending, as increasingly accessible by single-molecule techniques.

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In Vivo Thermodynamic Analysis of Repression with and without Looping in lac Constructs

Cited by 87 publications

References 0 publications

In Vitro Repression of the gal Promoters by GalR and HU Depends on the Proper Helical Phasing of the Two Operators

In Vitro Repression of the gal Promoters by GalR and HU Depends on the Proper Helical Phasing of the Two Operators

Transcriptional regulation by the numbers: models

Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity

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