Multivector fluorescence analysis of the xpt guanine riboswitch aptamer domain and the conformational role of guanine

Michael D. Brenner, Mary S. Scanlan, Michelle K. Nahas, Taekjip Ha, Scott K Silverman

Research output: Contribution to journalArticle

Abstract

Purine riboswitches are RNA regulatory elements that control purine metabolism in response to intracellular concentrations of the purine ligands. Conformational changes of the guanine riboswitch aptamer domain induced by guanine binding lead to transcriptional regulation of genes involved in guanine biosynthesis. The guanine riboswitch aptamer domain has three RNA helices designated P1, P2, and P3. An overall model for the Mg2+- and guanine-dependent relative orientations and dynamics of P1, P2, and P3 has not been reported, and the conformational role of guanine under physiologically relevant conditions has not been fully elucidated. In this study, an ensemble and single-molecule fluorescence resonance energy transfer (FRET) study was performed on three orthogonally labeled variants of the xpt guanine riboswitch aptamer domain. The combined FRET data support a model in which the unfolded state of the aptamer domain has a highly dynamic P2 helix that switches rapidly between two orientations relative to nondynamic Pl and P3. At ≤1 mM Mg 2+ (in the presence of a saturating level of guanine) or ≥ 1 mM Mg2+ (in the absence of guanine), the riboswitch starts to adopt a folded conformation in which loop - loop interactions lock P2 and P3 into place. At > 5 mM Mg2+, further compaction occurs in which P1 more closely approaches P3. Our data help to explain the biological role of guanine as stabilizing the globally folded aptamer domain conformation at physiologically relevant Mg2+ concentrations (≤1 mM), whereas in the absence of guanine, much higher Mg2+ concentrations are required to induce this folding event.

Original languageEnglish (US)
Pages (from-to)1596-1605
Number of pages10
JournalBiochemistry
Volume49
Issue number8
DOIs
StatePublished - Mar 2 2010

Fingerprint

Riboswitch
Guanine
Fluorescence
Fluorescence Resonance Energy Transfer
Conformations
RNA
Biosynthesis
Metabolism

ASJC Scopus subject areas

  • Biochemistry

Cite this

Multivector fluorescence analysis of the xpt guanine riboswitch aptamer domain and the conformational role of guanine. / Brenner, Michael D.; Scanlan, Mary S.; Nahas, Michelle K.; Ha, Taekjip; Silverman, Scott K.

In: Biochemistry, Vol. 49, No. 8, 02.03.2010, p. 1596-1605.

Research output: Contribution to journalArticle

Brenner, Michael D. ; Scanlan, Mary S. ; Nahas, Michelle K. ; Ha, Taekjip ; Silverman, Scott K. / Multivector fluorescence analysis of the xpt guanine riboswitch aptamer domain and the conformational role of guanine. In: Biochemistry. 2010 ; Vol. 49, No. 8. pp. 1596-1605.
@article{e2eb7a480f6d45049c956bcd3427e64d,
title = "Multivector fluorescence analysis of the xpt guanine riboswitch aptamer domain and the conformational role of guanine",
abstract = "Purine riboswitches are RNA regulatory elements that control purine metabolism in response to intracellular concentrations of the purine ligands. Conformational changes of the guanine riboswitch aptamer domain induced by guanine binding lead to transcriptional regulation of genes involved in guanine biosynthesis. The guanine riboswitch aptamer domain has three RNA helices designated P1, P2, and P3. An overall model for the Mg2+- and guanine-dependent relative orientations and dynamics of P1, P2, and P3 has not been reported, and the conformational role of guanine under physiologically relevant conditions has not been fully elucidated. In this study, an ensemble and single-molecule fluorescence resonance energy transfer (FRET) study was performed on three orthogonally labeled variants of the xpt guanine riboswitch aptamer domain. The combined FRET data support a model in which the unfolded state of the aptamer domain has a highly dynamic P2 helix that switches rapidly between two orientations relative to nondynamic Pl and P3. At ≤1 mM Mg 2+ (in the presence of a saturating level of guanine) or ≥ 1 mM Mg2+ (in the absence of guanine), the riboswitch starts to adopt a folded conformation in which loop - loop interactions lock P2 and P3 into place. At > 5 mM Mg2+, further compaction occurs in which P1 more closely approaches P3. Our data help to explain the biological role of guanine as stabilizing the globally folded aptamer domain conformation at physiologically relevant Mg2+ concentrations (≤1 mM), whereas in the absence of guanine, much higher Mg2+ concentrations are required to induce this folding event.",
author = "Brenner, {Michael D.} and Scanlan, {Mary S.} and Nahas, {Michelle K.} and Taekjip Ha and Silverman, {Scott K}",
year = "2010",
month = "3",
day = "2",
doi = "10.1021/bi9019912",
language = "English (US)",
volume = "49",
pages = "1596--1605",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "8",

}

TY - JOUR

T1 - Multivector fluorescence analysis of the xpt guanine riboswitch aptamer domain and the conformational role of guanine

AU - Brenner, Michael D.

AU - Scanlan, Mary S.

AU - Nahas, Michelle K.

AU - Ha, Taekjip

AU - Silverman, Scott K

PY - 2010/3/2

Y1 - 2010/3/2

N2 - Purine riboswitches are RNA regulatory elements that control purine metabolism in response to intracellular concentrations of the purine ligands. Conformational changes of the guanine riboswitch aptamer domain induced by guanine binding lead to transcriptional regulation of genes involved in guanine biosynthesis. The guanine riboswitch aptamer domain has three RNA helices designated P1, P2, and P3. An overall model for the Mg2+- and guanine-dependent relative orientations and dynamics of P1, P2, and P3 has not been reported, and the conformational role of guanine under physiologically relevant conditions has not been fully elucidated. In this study, an ensemble and single-molecule fluorescence resonance energy transfer (FRET) study was performed on three orthogonally labeled variants of the xpt guanine riboswitch aptamer domain. The combined FRET data support a model in which the unfolded state of the aptamer domain has a highly dynamic P2 helix that switches rapidly between two orientations relative to nondynamic Pl and P3. At ≤1 mM Mg 2+ (in the presence of a saturating level of guanine) or ≥ 1 mM Mg2+ (in the absence of guanine), the riboswitch starts to adopt a folded conformation in which loop - loop interactions lock P2 and P3 into place. At > 5 mM Mg2+, further compaction occurs in which P1 more closely approaches P3. Our data help to explain the biological role of guanine as stabilizing the globally folded aptamer domain conformation at physiologically relevant Mg2+ concentrations (≤1 mM), whereas in the absence of guanine, much higher Mg2+ concentrations are required to induce this folding event.

AB - Purine riboswitches are RNA regulatory elements that control purine metabolism in response to intracellular concentrations of the purine ligands. Conformational changes of the guanine riboswitch aptamer domain induced by guanine binding lead to transcriptional regulation of genes involved in guanine biosynthesis. The guanine riboswitch aptamer domain has three RNA helices designated P1, P2, and P3. An overall model for the Mg2+- and guanine-dependent relative orientations and dynamics of P1, P2, and P3 has not been reported, and the conformational role of guanine under physiologically relevant conditions has not been fully elucidated. In this study, an ensemble and single-molecule fluorescence resonance energy transfer (FRET) study was performed on three orthogonally labeled variants of the xpt guanine riboswitch aptamer domain. The combined FRET data support a model in which the unfolded state of the aptamer domain has a highly dynamic P2 helix that switches rapidly between two orientations relative to nondynamic Pl and P3. At ≤1 mM Mg 2+ (in the presence of a saturating level of guanine) or ≥ 1 mM Mg2+ (in the absence of guanine), the riboswitch starts to adopt a folded conformation in which loop - loop interactions lock P2 and P3 into place. At > 5 mM Mg2+, further compaction occurs in which P1 more closely approaches P3. Our data help to explain the biological role of guanine as stabilizing the globally folded aptamer domain conformation at physiologically relevant Mg2+ concentrations (≤1 mM), whereas in the absence of guanine, much higher Mg2+ concentrations are required to induce this folding event.

UR - http://www.scopus.com/inward/record.url?scp=77749304812&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77749304812&partnerID=8YFLogxK

U2 - 10.1021/bi9019912

DO - 10.1021/bi9019912

M3 - Article

C2 - 20108980

AN - SCOPUS:77749304812

VL - 49

SP - 1596

EP - 1605

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 8

ER -