The main goal of the proposed research is to understand the dynamics of transcriptional activation of human immunodeficiency virus type-1 (HIV-1) gene expression. HIV-1 encodes a transcriptional transactivator protein called Tat, which is expressed early in the viral life cycle and is absolutely required for viral replication and progression to disease. A regulatory element between +1 and +60 in tfie HIV-1 long ter repeat which is capable of forming a stable stem-loop structure designated TAR is critical for Tat function. In the absence of Tat, RNA polymerase 11 (pol 11) terminates transcription prematurely. Tat-TAR interactions convert pol II into its processive form and lead to the efficient production of full length viral transcripts. How does Tat increase RNA pol 11 processivity? What is the role of RNA-protein interactions in transactivation? A clear understanding of the novel mechanisms which control transcription elongation of HIV-1 and other genes is fundamental to understanding normal development as well as such diseases as cancer and AIDS. Our project has three specific aims.
Specific aim 1 : Isolation of elongation complexes. Experiments are proposed to develop a general method for isolating a homogeneous population of RNA polymerase II elongation complexes arrested at a DNA damage site. DNA damage will be introduced by a psoralen crosslink in DNA templates containing HIV-1 promoter. After performing transcription, elongation complexes will be purified and characterized.
Specific Aim 2 : RNA-protein interactions during transcription. Experiments are proposed to introduce modified nucleotfdes into the RNA sequences and perform photocrosslinking studies to identify proteins which interact with RNA.
Specific Aim 3 : Protein-protein interactions during transcription. Experiments are proposed to site-specifically modify protein side chains with photoactive groups and perform photocrosslinking studies to visualize protein-protein interactions.
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