TY - JOUR
T1 - Structural studies of DNA three-way junctions
AU - Leontis, Neocles B.
AU - Piotto, Martial E.
AU - Hills, Michael T.
AU - Malhotra, Arun
AU - Ouporov, Igor V.
AU - Nussbaum, Jean M.
AU - Gorenstein, David G.
N1 - Funding Information:
Research Corporation Grant C-2314 (to N. B. Leontis). The Purdue University Biochemical Magnetic Resonance Center, where most of the spectroscopic work was carried out, is supported by the NIH designated AIDS Research Center at Purdue (grant AI727713 to D. G. Gorenstein) and by the NSF Biological Facilities Center on Biomolecular NMR, Structure, and Design at Purdue (grants BBS 8614177 and 8714258 to D. G. Gorenstein from the Division of Biological Instrumentation). N. B. Leontis and A. Malhotra acknowledge the encouragement and support of Professor Stephen C. Harvey in the modeling studies. The authors thank Dr. Florence Lebreton for the 3D NOE-NOE pulse sequence and Dr. Dean Carlson for general technical assistance with NMR spectroscopy.
Funding Information:
This work was supported by U.S. Public Health Service Grants GM41454 (to N. B. Leontis), AI27744 (to D. G. Gorenstein), and GM34015 (to Stephen C. Harvey); by Ohio Board of Regents Research Challenge grants; by Petroleum Research Fund Grant 20871-GB4; and by 40 H. J. C. Berendsen, J. P. M. Postma, N. F. van Gunsteren, A. DiNola, and J. R. Haak, J. Chem. Phys. 81~ 3684 (1984). 41 I. V. Ouporov and N. B. Leontis, Biophys. J. 68, 266 (1995).
PY - 1995/1/1
Y1 - 1995/1/1
N2 - This chapter discusses the structural studies of DNA three-way junctions. Advances in the nuclear magnetic resonance (NMR) methods allow one to study increasingly complex molecular assemblages. The chapter describes the use of two- and three-dimensional homonuclear (proton) NMR, coupled with other experimental approaches, physical and chemical, to investigate the structure of DNA three-way junctions (TWJ) composed of three mutually complementary oligonucleotide strands. Synthetic TWJ oligonucleotide complexes serve as models of multibranch structures that occur frequently in biologically important nucleic acids, such as the ribosomal RNAs and the hammerhead ribozymes. Examination of the sequences, of naturally occurring TWJ, indicates that evolutionarily conserved unpaired bases are integral components of these structures. Using gel electrophoresis and UV melting techniques, the chapter shows that unpaired bases stabilize TWJ complexes. The TWJ that have been successfully studied by NMR have included two unpaired nucleotides in the junction region. The construction and refinement of the putative structural models based on the NMR data and the additional information obtained from chemical probing are also described in the chapter. Successful structure determination is greatly facilitated, by having at hand a molecular object, that exists in one predominant conformation or in a set of closely related conformational states.
AB - This chapter discusses the structural studies of DNA three-way junctions. Advances in the nuclear magnetic resonance (NMR) methods allow one to study increasingly complex molecular assemblages. The chapter describes the use of two- and three-dimensional homonuclear (proton) NMR, coupled with other experimental approaches, physical and chemical, to investigate the structure of DNA three-way junctions (TWJ) composed of three mutually complementary oligonucleotide strands. Synthetic TWJ oligonucleotide complexes serve as models of multibranch structures that occur frequently in biologically important nucleic acids, such as the ribosomal RNAs and the hammerhead ribozymes. Examination of the sequences, of naturally occurring TWJ, indicates that evolutionarily conserved unpaired bases are integral components of these structures. Using gel electrophoresis and UV melting techniques, the chapter shows that unpaired bases stabilize TWJ complexes. The TWJ that have been successfully studied by NMR have included two unpaired nucleotides in the junction region. The construction and refinement of the putative structural models based on the NMR data and the additional information obtained from chemical probing are also described in the chapter. Successful structure determination is greatly facilitated, by having at hand a molecular object, that exists in one predominant conformation or in a set of closely related conformational states.
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U2 - 10.1016/S0076-6879(95)61010-3
DO - 10.1016/S0076-6879(95)61010-3
M3 - Article
C2 - 8569495
AN - SCOPUS:0028834246
VL - 261
SP - 183
EP - 207
JO - Methods in Enzymology
JF - Methods in Enzymology
SN - 0076-6879
IS - C
ER -