Rational design of a super core promoter that enhances gene expression

Juven‐Gershon, Tamar; Cheng, Susan; Kadonaga, James T.

doi:10.1038/nmeth937

Cited by 190 publications

(210 citation statements)

References 20 publications

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“…Figure 1 depicts the experimental strategy and instrument design. Biotinylated, fluorescently labeled DNA templates containing a consensus Pol II promoter (super core) (Juven-Gershon et al 2006) were tethered to a biotinylated glass surface via streptavidin. Surface immobilization restricts the DNA Brownian motion and enables long-term tracking of molecular events on each DNA (Fig.…”

Section: Rationale and Methods Developmentmentioning

confidence: 99%

Transcription initiation by human RNA polymerase II visualized at single-molecule resolution

Revyakin¹,

Zhang²,

Coleman³

et al. 2012

Genes Dev.

109

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Forty years of classical biochemical analysis have identified the molecular players involved in initiation of transcription by eukaryotic RNA polymerase II (Pol II) and largely assigned their functions. However, a dynamic picture of Pol II transcription initiation and an understanding of the mechanisms of its regulation have remained elusive due in part to inherent limitations of conventional ensemble biochemistry. Here we have begun to dissect promoter-specific transcription initiation directed by a reconstituted human Pol II system at singlemolecule resolution using fluorescence video-microscopy. We detected several stochastic rounds of human Pol II transcription from individual DNA templates, observed attenuation of transcription by promoter mutations, observed enhancement of transcription by activator Sp1, and correlated the transcription signals with real-time interactions of holo-TFIID molecules at individual DNA templates. This integrated single-molecule methodology should be applicable to studying other complex biological processes.

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Section: Rationale and Methods Developmentmentioning

confidence: 99%

Transcription initiation by human RNA polymerase II visualized at single-molecule resolution

Revyakin¹,

Zhang²,

Coleman³

et al. 2012

Genes Dev.

109

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show abstract

“…Development 138 (12) several core promoter motifs) (Juven-Gershon et al, 2006), for enhancer trapping activity in Drosophila, Tribolium and Parhyale, but neither was found to work across the species tested (Schinko et al, 2010) (data not shown). Next, we searched for core promoters and splice acceptors among sequences that lie upstream of Parhyale hsp70 genes.…”

Section: Research Reportmentioning

confidence: 98%

A versatile strategy for gene trapping and trap conversion in emerging model organisms

et al. 2011

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SUMMARYGenetic model organisms such as Drosophila, C. elegans and the mouse provide formidable tools for studying mechanisms of development, physiology and behaviour. Established models alone, however, allow us to survey only a tiny fraction of the morphological and functional diversity present in the animal kingdom. Here, we present iTRAC, a versatile gene-trapping approach that combines the implementation of unbiased genetic screens with the generation of sophisticated genetic tools both in established and emerging model organisms. The approach utilises an exon-trapping transposon vector that carries an integrase docking site, allowing the targeted integration of new constructs into trapped loci. We provide proof of principle for iTRAC in the emerging model crustacean Parhyale hawaiensis: we generate traps that allow specific developmental and physiological processes to be visualised in unparalleled detail, we show that trapped genes can be easily cloned from an unsequenced genome, and we demonstrate targeting of new constructs into a trapped locus. Using this approach, gene traps can serve as platforms for generating diverse reporters, drivers for tissue-specific expression, gene knockdown and other genetic tools not yet imagined.

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“…Measuring transcription from single DNA molecules revealed that approximately 30% of the templates were transcribed when the strongest promoter was tested (an engineered "super core promoter" 21 ). A mutant version of this promoter showed lower template usage and addition of the activator Sp1, which could bind GC boxes upstream of the TATA box, increased template usage.…”

Section: Fluorescence Co-localization Can Be Used To Investigate Rnapmentioning

confidence: 99%

Single molecule studies of RNA polymerase II transcription in vitro

Horn¹,

Goodrich

Kugel³

2014

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Rational design of a super core promoter that enhances gene expression

Cited by 190 publications

References 20 publications

Transcription initiation by human RNA polymerase II visualized at single-molecule resolution

Transcription initiation by human RNA polymerase II visualized at single-molecule resolution

A versatile strategy for gene trapping and trap conversion in emerging model organisms

Single molecule studies of RNA polymerase II transcription in vitro

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