The HIV-1 Inducer of Short Transcripts Activates the Synthesis of 5,6-Dichloro-1-β-d-benzimidazole-resistant Short Transcripts in Vitro

Frank Pessler,Nouria Hernandez

doi:10.1074/jbc.273.9.5375

Abstract

The HIV-1 inducer of short transcripts (IST) is an unusual promoter element that activates the synthesis of short transcripts from the HIV-1 promoter as well as from heterologous promoters. While the DNA sequences constituting IST have been characterized in some detail, little is known about the biochemical mechanisms underlying IST activity. Here, we describe a cell-free transcription assay that faithfully reproduces the synthesis of IST-dependent HIV-1 short transcripts. As in vivo, formation of these short transcripts requires a functional IST element and is repressed in the presence of the viral trans-activator Tat. Short transcript and full-length transcript synthesis respond differently to variations in several reaction parameters, suggesting that the short and full-length transcripts are synthesized by transcription complexes with distinct biochemical properties. In particular, short transcript synthesis is resistant to the action of 5,6-dichloro-1-beta-D-benzimidazole, an inhibitor of transcript elongation. Formation of transcription complexes directed by the IST element may, therefore, not require the activity of a factor inhibited by 5, 6-dichloro-1-beta-D-benzimidazole, such as the TFIIH-associated or pTEFb kinases.

Highlights

The HIV-1 promoter directs the synthesis of two classes of RNAs that are initiated at the same transcription start site: full-length transcripts extending to the 3Ј-end of the transcription unit, and short, nonpolyadenylated RNAs of approximately 55– 65 nucleotides in length
While full-length transcripts are probably synthesized by elongation-competent transcription complexes capable of traversing the entire transcription unit, short transcripts have been hypothesized to result from a separate class of transcription complexes incapable of efficient transcript elongation [2, 4]
Mutations that debilitate the inducer of short transcripts (IST) element in vivo severely reduce the accumulation of short transcripts in vitro

Summary

Introduction

The HIV-1 promoter directs the synthesis of two classes of RNAs that are initiated at the same transcription start site: full-length transcripts extending to the 3Ј-end of the transcription unit, and short, nonpolyadenylated RNAs of approximately 55– 65 nucleotides in length. Synthesis of both the full-length and short HIV-1 transcripts was sensitive to the low concentrations of ␣-amanitin that typically inhibit RNA polymerase II transcription (lanes 2–5).

Results

Conclusion