The RNA element encoded by the trans-activation-responsive region of human immunodeficiency virus type 1 is functional when displaced downstream of the start of transcription.
The human immunodeficiency virus type 1 (HIV-1) trans-activator protein, Tat, specifically stimulates transcription from the viral long terminal repeat. Tat binds to an RNA stem-loop structure encoded by the trans-activation response region (TAR). To test whether TAR is functional when displaced downstream of the start of transcription, we assayed a series of templates carrying duplicated TAR elements in cell-free transcription systems. When the normally positioned TAR element (TAR-1) is inactivated by mutations in either the Tat binding site or the apical loop sequence, which acts as the binding site for a cellular factor, transactivation can be rescued by a wild-type TAR element placed downstream (TAR-2). The TAR-2 element is functional even when placed > 200 nt downstream of TAR-1. TAR complementation experiments have also shown that a functional TAR element requires both an intact Tat binding site and an intact apical loop sequence. For example, if TAR-1 carries a mutation in the loop element it cannot be rescued by a TAR-2 element carrying a mutation in the Tat binding site. Substitution mutations in TAR-1 show that the 5' half of TAR also encodes an essential DNA element which is required for efficient transcription initiation. These results strongly suggest that Tat and cellular cofactors which bind TAR RNA associate with the transcription complex during its transit through TAR.
Churcher
M J
MJ
Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
Lowe
A D
AD
Gait
M J
MJ
Karn
J
J
D001483:Base Sequence; D001665:Binding Sites; D002474:Cell-Free System; D015696:Gene Products, tat / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus