Fluorescence anisotropy: Rapid, quantitative assay for protein-DNA and protein-protein interaction

Heyduk, Tomasz; Ma, Yangmin; Tang, Hong; Ebright, Richard H.

doi:10.1016/s0076-6879(96)74039-9

Cited by 153 publications

(150 citation statements)

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“…We used fluorescence anisotropy to measure the kinetics of binding of purified Pum proteins to their cognate and noncognate RNA. Fluorescence anisotropy is widely used to investigate steady-state, dynamic equilibrium binding between protein and RNA (45)(46)(47).…”

Section: Methodsmentioning

confidence: 99%

Programmable RNA-binding protein composed of repeats of a single modular unit

Adamala

Martin-Alarcon

Boyden

2016

Proc. Natl. Acad. Sci. U.S.A.

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The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular protein architecture that targets unmodified RNA sequences in a programmable way. We report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which we call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. The specificity of such Pumby-RNA interactions was high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence. We validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. We further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems.

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Section: Methodsmentioning

confidence: 99%

Programmable RNA-binding protein composed of repeats of a single modular unit

Adamala

Martin-Alarcon

Boyden

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Interactions-The utility of using spectroscopy-based fluorescence assays to study protein-protein and protein-nucleic acid interactions has been amply documented (41), as has the use of EMSA (42). However, very few studies have actually utilized both methods under conditions wherein one can directly compare results.…”

Section: Emsa Measurement Of Tbp-dna and Tbp-taf130pmentioning

confidence: 99%

“…To accomplish this goal, we used fluorescently labeled DNA as previously (18,40) and also developed a fluorescent variant of TBP. Using these probes, we performed solution cuvette (41) and gel mobility shift assays (42), two complementary methods capable of precisely performing biophysical macromolecular interaction measurements. This is the first report combining these two techniques to study TBP dynamics.…”

mentioning

confidence: 99%

Fluorescence-based Analyses of the Effects of Full-length Recombinant TAF130p on the Interaction of TATA Box-binding Protein with TATA Box DNA

Banik

Beechem²,

Klebanow³

et al. 2001

Journal of Biological Chemistry

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We have used a combination of fluorescence anisotropy spectroscopy and fluorescence-based native gel electrophoresis methods to examine the effects of the transcription factor IID-specific subunit TAF130p (TAF145p) upon the TATA box DNA binding properties of TATA box-binding protein (TBP). Purified full-length recombinant TAF130p decreases TBP-TATA DNA complex formation at equilibrium by competing directly with DNA for binding to TBP. Interestingly, we have found that full-length TAF130p is capable of binding multiple molecules of TBP with nanomolar binding affinity. The biological implications of these findings are discussed.Eukaryotic DNA-dependent RNA polymerase II works in concert with the six general transcription factors (GTFs) 1 TFIIA, -B, -D, -E, -F, and -H to catalyze mRNA gene transcription (1). These components act either sequentially (2) or as a part of a holoenzyme complex (3, 4) to form a multicomponent preinitiation complex on promoter DNA. A highly conserved feature of most eukaryotic mRNA promoters is the TATA box element present 25 base pairs upstream from the transcription start site. TFIID, in combination with TFIIA and TFIIB, can recognize the TATA element and form a platform for subsequent preinitiation complex formation. The TATA box-binding protein (TBP), as its name suggests, is the protein within the 15-subunit TFIID holocomplex (5) that makes primary contact with the TATA element, though several TBP-associated factor (TAF) subunits comprising TFIID contribute to promoter binding (6 -8). Binding of TFIID to TATA elements is central to the control of transcription (9, 10).Recruiting TFIID to the promoter, a process thought to be mediated in part by direct activator-TFIID interactions, is probably a key and universal mechanism of gene activation (9, 10). However, the largest subunit of metazoan TFIID, which in Drosophila (d) or humans (h) exhibits an apparent molecular mass of ϳ250 kDa, termed d-or hTAF II 250, respectively (herein termed d/hTAF250p), also contains a number of intrinsic enzymatic activities including histone acetyltransferase (11), protein kinase (12), and ubiquitin activating/conjugating activity (13). Each of these activities could be targets for transactivators, and mutation of histone acetyltransferase, protein kinase, or ubiquitin activating/conjugating activity domains decreases transcription of subsets of genes in vivo (13-15).The yeast ortholog of d/hTAF250, TAF130p, is encoded by a single-copy essential gene (TAF130/TAF145; Refs. 16 and 17). Like its metazoan counterparts, the yeast protein, TAF130p, contains a histone acetyltransferase domain as well as a number of other essential sequences (18, 19) whose exact functions remain to be defined. One highly conserved and important element found in this (family of) TAF are domains capable of directly binding the TBP subunit of TFIID (17, 18, 20 -24). Both Drosophila TAF250p and yeast TAF130p appear to contain at least two TBP binding domains (18,23,25): a high affinity N-terminal TBP binding domain and a less well de...

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“…This assay, although highly informative, identifies only the best binding sequences, whereas the less optimal, and often biologically relevant, sequences are missed. Other commonly used biochemical or biophysical approaches are labor-intensive and can be used only to study a limited set of sequence variants (5)(6)(7)(8)(9)(10). Medium-throughput microarrays have also been developed in which duplex DNA molecules are immobilized on surfaces and protein binding is detected by surface plasmon resonance (11) or fluorescence (12,13).…”

mentioning

confidence: 99%

Defining the sequence-recognition profile of DNA-binding molecules

Warren

Kratochvil

Hauschild

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

222

238

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Determining the sequence-recognition properties of DNA-binding proteins and small molecules remains a major challenge. To address this need, we have developed a high-throughput approach that provides a comprehensive profile of the binding properties of DNA-binding molecules. The approach is based on displaying every permutation of a duplex DNA sequence (up to 10 positional variants) on a microfabricated array. The entire sequence space is interrogated simultaneously, and the affinity of a DNA-binding molecule for every sequence is obtained in a rapid, unbiased, and unsupervised manner. Using this platform, we have determined the full molecular recognition profile of an engineered small molecule and a eukaryotic transcription factor. The approach also yielded unique insights into the altered sequence-recognition landscapes as a result of cooperative assembly of DNA-binding molecules in a ternary complex. Solution studies strongly corroborated the sequence preferences identified by the array analysis.chemical genomics ͉ ligand-DNA recognition

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Fluorescence anisotropy: Rapid, quantitative assay for protein-DNA and protein-protein interaction

Cited by 153 publications

References 21 publications

Programmable RNA-binding protein composed of repeats of a single modular unit

Programmable RNA-binding protein composed of repeats of a single modular unit

Fluorescence-based Analyses of the Effects of Full-length Recombinant TAF130p on the Interaction of TATA Box-binding Protein with TATA Box DNA

Defining the sequence-recognition profile of DNA-binding molecules

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