Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.

Abstract

The human immunodeficiency virus (HIV-1)-encoded trans-activator (tat) increases HIV gene expression and replication. Previously, we demonstrated that tat facilitates elongation of transcription through the HIV-1 long terminal repeat (LTR) and that short transcripts corresponding to prematurely terminated RNA are released and accumulate in the absence of tat. Here, using a transient expression assay, we tested clustered and compensatory mutations, as wall as 3' deletions, in the trans-acting responsive region (tar) and observed that the primary sequence in the loop and secondary structure in the stem of the stem-loop in tar are required for trans-activation by tat. Insertions in the 5' region of tar revealed that tar must be near the site of HIV-1 initiation of transcription for trans-activation by tat. Deletions (3') and an insertion in tar demonstrated that an intact stem-loop is required for the recovery of prematurely terminated transcripts. Short and full-length transcripts were observed also with HIV type 2 (HIV-2) in the absence and presence of tat, respectively. We conclude that an intact stem-loop in tar is essential for trans-activation by tat and that initiation of transcription by HIV-1 promoter factors and elongation of transcription by tat are coupled.

Genetic Analysis of the Structure and Function of 7SK Small Nuclear Ribonucleoprotein (snRNP) in Cells

“…In the absence of Tat, RNAPII transcribes up to 150 nucleotides past the transcriptional start site on the HIV LTR (50). Supporting this observation, recent genome-wide analyses revealed that pausing sites distribute over an extended distance after initiation (51).…”

Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes

“…Deletion of a 70 bp region containing this NRE increased promoter activity by 5-6-fold, and also suppressed the heterologous TK promoter in an orientation-independent but position-dependent manner. These observations led to the hypothesis that the silencer affects transcriptional elongation by premature termination [102], as has been identified in the c-myc [103], c-myb [104], HIV-1 and HIV-2 promoters [105] or by pausing RNA pol II as occurs in the Drosophila heat-shock protein 70 (HSP70) gene [106]. However, further evidence is required to substantiate this hypothesis.…”

Regulation of eukaryotic gene expression by transcriptional attenuation.

“…There are, however, clear differences between the TATand N-mediated antitermination processes. The X nut site will function as a discrete element when moved away from its cognate promoter, whereas TAT-mediated antitermination requires TAR to be located immediately adjacent to the HIV promoter (Selby et al, 1989), and it also remains unclear whether TAT acts to overcome specific termination signals in the HIV TAR region in a manner analogous to the antitermination of the prokaryotic terminators. Most of the recent evidence suggests that in the absence of TAT, LTR-initiated transcription terminates at many, possibly random, downstream sites (Lapsia et al, 1989;Marciniak and Sharp, 1991;Kessler and Mathew, 1992).…”