HIV-1 TAR RNA Enhances the Interaction between Tat and Cyclin T1

Jing Zhang,Natarajan Tamilarasu,Seongwoo Hwang,Mitchell E Garber,Ikramul Huq,Katherine A Jones,Tariq M Rana

doi:10.1074/jbc.m006804200

Abstract

Human immunodeficiency virus, type 1 (HIV-1), Tat activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex, P-TEFb. Binding of Tat to CycT1 induces cooperative binding of the P-TEFb complex onto nascent HIV-1 TAR RNA. Here the specific interaction between Tat protein, human cyclin T1, and HIV-1 TAR RNA was analyzed by fluorescence resonance energy transfer, using fluorescein-labeled TAR RNA and a rhodamine-labeled Tat protein synthesized through solid-phase chemistry. We find that CycT1 remodels the structure of Tat to enhance its affinity for TAR RNA and that TAR RNA further enhances the interaction between Tat and CycT1. We conclude that TAR RNA nucleates the formation of the Tat.P-TEFb complex through an induced fit mechanism.

Highlights

The human immunodeficiency virus (HIV-1)1 encodes a transcriptional activator protein, Tat, that increases the processivity of RNA polymerase II
Tat activates transcription through binding to the upper stem and bulge region of TAR, a structured element in the nascent viral RNA, and controls a DRB-sensitive step early in RNAPII transcription elongation that results in hyperphosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II
We developed a fluorescence resonance energy transfer (FRET) system containing TAR RNA and Tat protein uniquely labeled with donor and acceptor dye molecules (Fig. 1)

Summary

Introduction

The human immunodeficiency virus (HIV-1)1 encodes a transcriptional activator protein, Tat, that increases the processivity of RNA polymerase II (for reviews see Refs. 1–3). We developed a fluorescence resonance energy transfer (FRET) system containing TAR RNA and Tat protein uniquely labeled with donor and acceptor dye molecules (Fig. 1).

Results

Conclusion