Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.
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 well 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.
Selby
M J
MJ
Howard Hughes Medical Institute, Department of Medicine, University of California, San Francisco 94143.
Bain
E S
ES
Luciw
P A
PA
Peterlin
B M
BM
D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D002872:Chromosome Deletion; D005786:Gene Expression Regulation; D015497:HIV-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012313:RNA / Q000032:analysis / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D012260:Ribonucleases / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D012313:RNA; D015500:Chloramphenicol O-Acetyltransferase; D012260:Ribonucleases