DETERMINATION OF 3D STRUCTURE OF RNA OLIGONUCLEOTIDE BY NMR SPECTROSCOPY

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The SIR provided 13C15 N ASI cells; 20g [U- "C, "N]AS 1 Cells; 50g The genome of HIV codes for two proteins, Tat and Rev, which are essential regulators of transcription. Rev binds to specific RNA sequence called the Rev Responsive Element (RRE), which is found in the HIV genome and regulates the cytoplasmic appearance of unspliced or singly spliced mRNAs which encode the structural proteins gag, pol, and env. Rev has been proposed to have a role both in regulation of splicing and in transport of the RNA to the cytoplasm. Chemical and RNAse Protection experiments have shown that a 66 nucleotide fragment, domain II of the RRE, is necessary and sufficient for high affinity binding of Rev in in vitro and that domain H alone is sufficient for a detectable Rev responsiveness in vivo. The core Rev binding element within the RRE and the critical bases or base-base interactions have recently been more precisely defined by an interactive in vitro genetic selection as well as other techniques. RNA oligonucleotides 30-35 nucleotides long containing the core binding element were shown to bind Rev with wild type affinity. Other studies have shown that a 17 amino acid arginine rich peptide from Rev binds RRE at multiple sites and specifically inhibits splicing in vitro. A single peptide specifically binds in the core element of the RRE. We propose the us multidimensional, multinuclear NMR spectroscopy to elucidate the structural features of the RRE that are important in REV recognition and binding. 13C and 15N labeled samples of RNA synthesized using labeled NTPs isolated from the RNA of methanolotrophic bacterial. These labeled samples will be used in NMR studies of the structure of RNA and RNA-Rev peptide complexes. Various derivatives of the consensus RNA sequence will also be studied in order to further define the structural requirements for Rev.