How ribonucleic acid (RNA) molecules fold into tertiary structures, how proteins recognize specific RNA structures, and how protein binding is involved in RNA functions are central questions to a number of biological processes. The broad goal of this proposal is to elucidate the high-resolution structures and mechanistic principles that underlie the formation of a particular RNA structural fold (the loop-loop """"""""kissing"""""""" motif) in three different and unique systems using multi- dimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy and other biophysical methods. RNA loop-loop """"""""kissing"""""""" interactions form principally via base-pairing between loops and/or bulges with complementary nucleotide sequences and are the critical intermediate structures involved in the antisense regulation of a variety of cellular processes in bacteria. These interactions are also involved in the proper folding of certain mRNA, rRNA and catalytic RNA structures. As an important element in dimerization of genomic retroviral RNA, the loop-loop """"""""kissing"""""""" structure is also a viable target for anti-viral drugs.
The specific aims of this research are: 1) to determine the structural basis for specific interaction in two novel RNA loop-loop """"""""kissing"""""""" complexes: a homodimeric loop-loop """"""""kissing"""""""" complex formed by an RNA stem-loop derived from the dimerization initiation site (DIS) in HIV-1 genomic RNA and a loop-loop """"""""kissing"""""""" complex formed by RNA stem-loops derived from the mRNA CopA and antisense CopT repressor transcripts involved in R1 plasmid replication control; and 2) to determine the structural basis for the specific recognition of a loop- loop """"""""kissing"""""""" complex derived from the ColE1 replication control system by the protein Rom. NMR spectroscopic structural and dynamical studies will be complemented by stop-flow kinetic, thermal melting and gel electrophoresis experiments designed to assay thermodynamic stability and association/dissociation kinetics of wild type and mutant loop-loop """"""""kissing"""""""" complexes. New NMR methods, specifically applicable to 13C, 15N and/or 2H labeled RNA and RNA-protein complexes of sizes (10 kDa - 26 kDa), will also be designed which will provide higher precision and sensitivity than available with existing technology and which will access global long-range structural restraints currently unavailable for RNA molecules.
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