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Probing the Rate-Limiting Step for Intramolecular Transfer of a Transcription Factor between Specific Sites on the Same DNA Molecule by (15)N(z)-Exchange NMR Spectroscopy
[Image: see text] The kinetics of translocation of the homeodomain transcription factor HoxD9 between specific sites of the same or opposite polarities on the same DNA molecule have been studied by (15)N(z)-exchange NMR spectroscopy. We show that exchange occurs by two facilitated diffusion mechanis...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical
Society
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210153/ https://www.ncbi.nlm.nih.gov/pubmed/25253516 http://dx.doi.org/10.1021/ja5081585 |
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author | Ryu, Kyoung-Seok Tugarinov, Vitali Clore, G. Marius |
author_facet | Ryu, Kyoung-Seok Tugarinov, Vitali Clore, G. Marius |
author_sort | Ryu, Kyoung-Seok |
collection | PubMed |
description | [Image: see text] The kinetics of translocation of the homeodomain transcription factor HoxD9 between specific sites of the same or opposite polarities on the same DNA molecule have been studied by (15)N(z)-exchange NMR spectroscopy. We show that exchange occurs by two facilitated diffusion mechanisms: a second-order intermolecular exchange reaction between specific sites located on different DNA molecules without the protein dissociating into free solution that predominates at high concentrations of free DNA, and a first-order intramolecular process involving direct transfer between specific sites located on the same DNA molecule. Control experiments using a mixture of two DNA molecules, each possessing only a single specific site, indicate that transfer between specific sites by full dissociation of HoxD9 into solution followed by reassociation is too slow to measure by z-exchange spectroscopy. Intramolecular transfer with comparable rate constants occurs between sites of the same and opposing polarity, indicating that both rotation-coupled sliding and hopping/flipping (analogous to geminate recombination) occur. The half-life for intramolecular transfer (0.5–1 s) is many orders of magnitude larger than the calculated transfer time (1–100 μs) by sliding, leading us to conclude that the intramolecular transfer rates measured by z-exchange spectroscopy represent the rate-limiting step for a one-base-pair shift from the specific site to the immediately adjacent nonspecific site. At zero concentration of added salt, the intramolecular transfer rate constants between sites of opposing polarity are smaller than those between sites of the same polarity, suggesting that hopping/flipping may become rate-limiting at very low salt concentrations. |
format | Online Article Text |
id | pubmed-4210153 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-42101532015-09-25 Probing the Rate-Limiting Step for Intramolecular Transfer of a Transcription Factor between Specific Sites on the Same DNA Molecule by (15)N(z)-Exchange NMR Spectroscopy Ryu, Kyoung-Seok Tugarinov, Vitali Clore, G. Marius J Am Chem Soc [Image: see text] The kinetics of translocation of the homeodomain transcription factor HoxD9 between specific sites of the same or opposite polarities on the same DNA molecule have been studied by (15)N(z)-exchange NMR spectroscopy. We show that exchange occurs by two facilitated diffusion mechanisms: a second-order intermolecular exchange reaction between specific sites located on different DNA molecules without the protein dissociating into free solution that predominates at high concentrations of free DNA, and a first-order intramolecular process involving direct transfer between specific sites located on the same DNA molecule. Control experiments using a mixture of two DNA molecules, each possessing only a single specific site, indicate that transfer between specific sites by full dissociation of HoxD9 into solution followed by reassociation is too slow to measure by z-exchange spectroscopy. Intramolecular transfer with comparable rate constants occurs between sites of the same and opposing polarity, indicating that both rotation-coupled sliding and hopping/flipping (analogous to geminate recombination) occur. The half-life for intramolecular transfer (0.5–1 s) is many orders of magnitude larger than the calculated transfer time (1–100 μs) by sliding, leading us to conclude that the intramolecular transfer rates measured by z-exchange spectroscopy represent the rate-limiting step for a one-base-pair shift from the specific site to the immediately adjacent nonspecific site. At zero concentration of added salt, the intramolecular transfer rate constants between sites of opposing polarity are smaller than those between sites of the same polarity, suggesting that hopping/flipping may become rate-limiting at very low salt concentrations. American Chemical Society 2014-09-25 2014-10-15 /pmc/articles/PMC4210153/ /pubmed/25253516 http://dx.doi.org/10.1021/ja5081585 Text en Copyright © 2014 American Chemical Society |
spellingShingle | Ryu, Kyoung-Seok Tugarinov, Vitali Clore, G. Marius Probing the Rate-Limiting Step for Intramolecular Transfer of a Transcription Factor between Specific Sites on the Same DNA Molecule by (15)N(z)-Exchange NMR Spectroscopy |
title | Probing
the Rate-Limiting Step for Intramolecular
Transfer of a Transcription Factor between Specific Sites on the Same
DNA Molecule by (15)N(z)-Exchange
NMR Spectroscopy |
title_full | Probing
the Rate-Limiting Step for Intramolecular
Transfer of a Transcription Factor between Specific Sites on the Same
DNA Molecule by (15)N(z)-Exchange
NMR Spectroscopy |
title_fullStr | Probing
the Rate-Limiting Step for Intramolecular
Transfer of a Transcription Factor between Specific Sites on the Same
DNA Molecule by (15)N(z)-Exchange
NMR Spectroscopy |
title_full_unstemmed | Probing
the Rate-Limiting Step for Intramolecular
Transfer of a Transcription Factor between Specific Sites on the Same
DNA Molecule by (15)N(z)-Exchange
NMR Spectroscopy |
title_short | Probing
the Rate-Limiting Step for Intramolecular
Transfer of a Transcription Factor between Specific Sites on the Same
DNA Molecule by (15)N(z)-Exchange
NMR Spectroscopy |
title_sort | probing
the rate-limiting step for intramolecular
transfer of a transcription factor between specific sites on the same
dna molecule by (15)n(z)-exchange
nmr spectroscopy |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210153/ https://www.ncbi.nlm.nih.gov/pubmed/25253516 http://dx.doi.org/10.1021/ja5081585 |
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