The human immunodeficiency virus long terminal repeat includes a specialised initiator element which is required for Tat-responsive transcription.
The effects of mutations in human immunodeficiency virus type-1 (HIV-1) long terminal repeat on initiation and on Tat-mediated trans-activation were studied using cell-free transcription assays. All the elements that are necessary for efficient transcription initiation in vitro are included in the core promoter. This region contains three tandem Sp1 binding sites, a TATA element and an initiator (INR) sequence. Although the HIV-1 INR element overlaps the trans-activation response region (TAR), it forms an integral part of the promoter. The HIV-1 INR element was characterised in detail using a template that carries a complete HIV-1 promoter and a displaced TAR RNA element. The results demonstrate that the sequence G+1GGTCT is essential for HIV-1 INR function. RNase protection experiments show that Tat acts exclusively to stimulate transcriptional elongation. Mutations in the core promoter elements reduce initiation rates dramatically but do not block Tat activity. For each mutation studied, the total level of transcription in the presence of Tat is proportional to the rate of initiation in the absence of Tat. Furthermore the rate of initiation remains constant in the presence or absence of Tat. We conclude that the elements of the HIV-1 core promoter act in concert to simulate initiation. By contrast, Tat acts independently of the core promoter elements and stimulates elongation. The data strongly suggest that Tat is recruited to the elongating transcription complex during its transit through TAR.
Rittner
K
K
MRC Laboratory of Molecular Biology, Cambridge, UK.
Churcher
M J
MJ
Gait
M J
MJ
Karn
J
J
D001483:Base Sequence; D002474:Cell-Free System; D004279:DNA, Viral; D015696:Gene Products, tat / Q000502:physiology*; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000235:genetics*; D054322:tat Gene Products, Human Immunodeficiency Virus
D004279:DNA, Viral; D015696:Gene Products, tat; D016328:NF-kappa B; D012333:RNA, Messenger; D012367:RNA, Viral; D016329:Sp1 Transcription Factor; D054322:tat Gene Products, Human Immunodeficiency Virus