8] Structural studies of DNA three-way junctions

Abstract

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.