TY - JOUR
T1 - Formation of Tertiary Interactions during rRNA GTPase Center Folding
AU - Rau, Michael J.
AU - Welty, Robb
AU - Tom Stump, W.
AU - Hall, Kathleen B.
N1 - Funding Information:
2AP-RNA and 15 N-RNA were synthesized by Agilent laboratories, and we thank Dr. Laurakay Bruhn, Dr. Jeff Sampson, and Dr. Doug Dellinger. We thank Professor Roberto Galletto for use of his stopped-flow spectrometer. This work was supported by a grant to K.B.H. ( National Institutes of Health R01 GM098102 ) and a gift from Agilent.
Publisher Copyright:
© 2015 Elsevier Ltd. All rights reserved.
PY - 2015/8/17
Y1 - 2015/8/17
N2 - The 60-nt GTPase center (GAC) of 23S rRNA has a phylogenetically conserved secondary structure with two hairpin loops and a 3-way junction. It folds into an intricate tertiary structure upon addition of Mg2 + ions, which is stabilized by the L11 protein in cocrystal structures. Here, we monitor the kinetics of its tertiary folding and Mg2 +-dependent intermediate states by observing selected nucleobases that contribute specific interactions to the GAC tertiary structure in the cocrystals. The fluorescent nucleobase 2-aminopurine replaced three individual adenines, two of which make long-range stacking interactions and one that also forms hydrogen bonds. Each site reveals a unique response to Mg2 + addition and temperature, reflecting its environmental change from secondary to tertiary structure. Stopped-flow fluorescence experiments revealed that kinetics of tertiary structure formation upon addition of MgCl2 are also site specific, with local conformational changes occurring from 5 ms to 4 s and with global folding from 1 to 5 s. Site-specific substitution with 15N-nucleobases allowed observation of stable hydrogen bond formation by NMR experiments. Equilibrium titration experiments indicate that a stable folding intermediate is present at stoichiometric concentrations of Mg2 + and suggest that there are two initial sites of Mg2 + ion association.
AB - The 60-nt GTPase center (GAC) of 23S rRNA has a phylogenetically conserved secondary structure with two hairpin loops and a 3-way junction. It folds into an intricate tertiary structure upon addition of Mg2 + ions, which is stabilized by the L11 protein in cocrystal structures. Here, we monitor the kinetics of its tertiary folding and Mg2 +-dependent intermediate states by observing selected nucleobases that contribute specific interactions to the GAC tertiary structure in the cocrystals. The fluorescent nucleobase 2-aminopurine replaced three individual adenines, two of which make long-range stacking interactions and one that also forms hydrogen bonds. Each site reveals a unique response to Mg2 + addition and temperature, reflecting its environmental change from secondary to tertiary structure. Stopped-flow fluorescence experiments revealed that kinetics of tertiary structure formation upon addition of MgCl2 are also site specific, with local conformational changes occurring from 5 ms to 4 s and with global folding from 1 to 5 s. Site-specific substitution with 15N-nucleobases allowed observation of stable hydrogen bond formation by NMR experiments. Equilibrium titration experiments indicate that a stable folding intermediate is present at stoichiometric concentrations of Mg2 + and suggest that there are two initial sites of Mg2 + ion association.
KW - 2-aminopurine
KW - Mg-dependent RNA folding
KW - NMR
KW - RNA folding kinetics
KW - stopped-flow fluorescence
UR - http://www.scopus.com/inward/record.url?scp=84939261130&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2015.07.013
DO - 10.1016/j.jmb.2015.07.013
M3 - Article
C2 - 26210661
AN - SCOPUS:84939261130
SN - 0022-2836
VL - 427
SP - 2799
EP - 2815
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 17
ER -