Lac repressor-operator interaction: DNA length dependence

Khoury, Anastasia M.; Lee, Hyun Joo; Lillis, Marcella; Lu, Ponzy

doi:10.1016/0167-4781(90)90120-q

Cited by 25 publications

(19 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Experimental artifacts may greatly overshadow actual values, which should come as no surprise in the case of lac repressor binding to operator DNA where the published binding constant has changed 33-fold as experimental techniques have changed. The difficulty in in vitro measurements is well known in the field as is evidenced by the large consideration given to differences in buffer conditions (Ha et al, 1992), DNA length (Khoury et al, 1990), and even hydrostatic pressure (Royer, Chakerian, & Matthews, 1990). Techniques such as gel filtration or nitrocellulose filter binding assays are excellent at differentiating binding strength between point mutants; they are limited in comparison with in vivo results.…”

Section: Discussionmentioning

confidence: 99%

In vitro transcription accurately predicts lac repressor phenotype in vivo in Escherichia coli

Sochor

2014

Preprint

View full text Add to dashboard Cite

A multitude of studies have looked at the in vivo and in vitro behavior of the lac repressor binding to DNA and effector molecules in order to study transcriptional repression, however these studies are not always reconcilable. Here we use in vitro transcription to directly mimic the in vivo system in order to build a self consistent set of experiments to directly compare in vivo and in vitro genetic repression. A thermodynamic model of the lac repressor binding to operator DNA and effector is used to link DNA occupancy to either normalized in vitro mRNA product or normalized in vivo fluorescence of a regulated gene, YFP. An accurate measurement of repressor, DNA and effector concentrations were made both in vivo and in vitro allowing for direct modeling of the entire thermodynamic equilibrium. In vivo repression profiles are accurately predicted from the given in vitro parameters when molecular crowding is considered. Interestingly, our measured repressor-operator DNA affinity differs significantly from previous in vitro measurements. The literature values are unable to replicate in vivo binding data. We therefore conclude that the repressor-DNA affinity is much weaker than previously thought. This finding would suggest that in vitro techniques that are specifically designed to mimic the in vivo process may be necessary to replicate the native system.

show abstract

Section: Discussionmentioning

confidence: 99%

In vitro transcription accurately predicts lac repressor phenotype in vivo in Escherichia coli

Sochor

2014

Preprint

View full text Add to dashboard Cite

show abstract

“…A number of biochemical assays have developed indirect evidence that lends support to the sliding mechanism (41)(42)(43). However, only single-molecule experiments permit direct observation of the trajectories of individual RNAP molecules during the promoter-search process, providing a unique window into this phase of initiation.…”

Section: Initiation Promoter Searchmentioning

confidence: 99%

Single-Molecule Studies of RNA Polymerase: Motoring Along

Herbert

Greenleaf

Block

2008

Annu. Rev. Biochem.

187

141

View full text Add to dashboard Cite

Single-molecule techniques have advanced our understanding of transcription by RNA polymerase. A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNA polymerase identifies a promoter; initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNA polymerase, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates.

show abstract

“…This hypothesis assumes that the initial binding to a repeat of shadow sites is weak and that the transcription factor quickly slides or jumps to stronger neighboring sites. The possibility of such lateral diffusion for transcription factors on DNA has been widely discussed in the literature (Berg et al 1981;Berg and von Hippel 1985;Khory et al 1990).…”

Section: Bstf Arrangements and Role Of Tandem Clustersmentioning

confidence: 99%

Extraction of Functional Binding Sites from Unique Regulatory Regions: The Drosophila Early Developmental Enhancers

Papatsenko¹,

Makeev

Lifanov³

et al. 2002

Genome Res.

View full text Add to dashboard Cite

The early developmental enhancers of Drosophila melanogaster comprise one of the most sophisticated regulatory systems in higher eukaryotes. An elaborate code in their DNA sequence translates both maternal and early embryonic regulatory signals into spatial distribution of transcription factors. One of the most striking features of this code is the redundancy of binding sites for these transcription factors (BSTF). Using this redundancy, we explored the possibility of predicting functional binding sites in a single enhancer region without any prior consensus/matrix description or evolutionary sequence comparisons. We developed a conceptually simple algorithm, Scanseq, that employs an original statistical evaluation for identifying the most redundant motifs and locates the position of potential BSTF in a given regulatory region. To estimate the biological relevance of our predictions, we built thorough literature-based annotations for the best-known Drosophila developmental enhancers and we generated detailed distribution maps for the most robust binding sites. The high statistical correlation between the location of BSTF in these experiment-based maps and the location predicted in silico by Scanseq confirmed the relevance of our approach. We also discuss the definition of true binding sites and the possible biological principles that govern patterning of regulatory regions and the distribution of transcriptional signals.

show abstract

Lac repressor-operator interaction: DNA length dependence

Cited by 25 publications

References 32 publications

In vitro transcription accurately predicts lac repressor phenotype in vivo in Escherichia coli

In vitro transcription accurately predicts lac repressor phenotype in vivo in Escherichia coli

Single-Molecule Studies of RNA Polymerase: Motoring Along

Extraction of Functional Binding Sites from Unique Regulatory Regions: The Drosophila Early Developmental Enhancers

Contact Info

Product

Resources

About