Binding Characteristics of TAR RNA and Tat‐Cyc peptide from the Human Immunodeficiency Virus

Abstract

The trans‐activation of transcription (Tat) peptide and its binding to the trans‐activating response element (TAR) RNA are observable in the human immunodeficiency virus (HIV). The interaction between the TAR RNA and the Tat peptide immensely increases the transcription efficiency of HIV by preventing cells from inducing apoptosis. Optimal binding of Tat to TAR requires the presence of a host protein, cyclin T1 to allow phosphorylation of the stalled RNA polymerase II. To study the interaction between trans‐activation of transcription (Tat) peptide and the trans‐activating response element (TAR) RNA of HIV, the cDNA for a fusion of the Tat peptide with a cyclin T1 peptide (Tat‐Cyc) and TAR RNA were both cloned separately by PCR‐assisted gene assembly using overlapping primers. The PCR products were initially ligated into the pGEM‐T vector (Promega). The cDNA for the TAR RNA was then subcloned into the BamHI and EcoRI restriction sites of vector pUC18 and will be transcribed via T7 RNA polymerase. The Tat‐cyc peptide was subcloned into the expression vector pSUMO‐Star (LifeSensors) at the BsaI and Xho I restriction sites. Once both the TAR RNA and Tat‐cyc peptides have been purified, the binding characteristics of the two will be analyzed and observed using Isothermal Titration Calorimetry (ITC) and gel shift binding assays. This TAR RNA and TAT peptide recognition study could promote the development of therapeutic agents to block or inhibit the viral activation and transcription of HIV.