Transcription- the fundamental process by which genetic information is expressed--defines a central problem in structural molecular biology. This propsal focuses on novel mechanisms of gene regulation. NMR methods will be used to investigate complementary structural motifs related to transcriptional elongation. These structures functionally intertwined are of broad relevance to cancer biology and AIDS.
Aims 1 A and 1B. Protein-RNA Recognition in a Viral Transcriptional Program Gene regulation in mammalian immunodeficency viruses (including HIV- 1) is medicated by specific RNA binding proteins Tat and Rev, which bind to RNA enhancer elements in the nascent message. Required for virulence, such elongation factors share an arginine-rich RNA-binding motif. A classical model is provided by the N protein of phage lambda. Because this model is supported by a wealth of classical and molecular genetics, we propose to determine the structure of a specific N peptide- RNA complex (Aim 1A) and to dissect this structure by mutagenesis (Aim 1B). Preliminary results suggest that its comparison with HIV-1 will deepen our understanding of protein-RNA recognition and its role in viral gene regulation. Structural effects of mutations will be correlate with functional changes in the transcriptional program of the phage.
Aims 2 A and 2B. A Novel Zn Ribbon in a Human Elongation Factor and RNA Polymerase. TFIIS, a conserved eukaryotic transcriptional elongation factor, contains a novel nucleic-acid binding domain. TFIIS is required for antitermination at pause sites in nuclear proto-oncogenes and can act in synergy with HIV-1Tat. To relate strucure and function, we will determine the strucures of mutant human domains associated with defects in antitermination and RNA recognition (Aim 2A). A putative Zn ribbon is conserved in a functionally related subnunit of eukaryotic and archeal RNA polymerases (RNAP subunit 9 ). Because the structure of neither RNAP is known, we propose to determine the structure of this subunit from Thermococcus celer (Aim 2B). Together, our Aims offer the exciting possibility of applying structural methods to a central problem in molecular biology, the regulation of transcriptional elongation. Further, use of hyperthermophilic archeal proteins suggests a general strategy toward high-resolution NMR study of conserved RNAP subunits. The systems under study are of broad biological interest with application to cancer biology and AIDS.
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