Transfer of Tat and release of TAR RNA during the activation of the human immunodeficiency virus type-1 transcription elongation complex.
The HIV-1 trans-activator protein, Tat, is a potent activator of transcriptional elongation. Tat is recruited to the elongating RNA polymerase during its transit through the trans-activation response region (TAR) because of its ability to bind directly to TAR RNA expressed on the nascent RNA chain. We have shown that transcription complexes that have acquired Tat produce 3-fold more full-length transcripts than complexes not exposed to Tat. Western blotting experiments demonstrated that Tat is tightly associated with the paused polymerases. To determine whether TAR RNA also becomes attached to the transcription complex, DNA oligonucleotides were annealed to the nascent chains on the arrested complexes and the RNA was cleaved by RNase H. After cleavage, the 5' end of the nascent chain, carrying TAR RNA, is quantitatively removed, but the 3' end of the transcript remains associated with the transcription complex. Even after the removal of TAR RNA, transcription complexes that have been activated by Tat show enhanced processivity. We conclude that Tat, together with cellular co-factors, becomes attached to the transcription complex and stimulates processivity, whereas TAR RNA does not play a direct role in the activation of elongation and is used simply to recruit Tat and cellular co-factors.
Keen
N J
NJ
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Churcher
M J
MJ
Karn
J
J
D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D029968:Escherichia coli Proteins; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D056968:Lac Repressors; D008957:Models, Genetic; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D015696:Gene Products, tat; D056968:Lac Repressors; D012367:RNA, Viral; D012097:Repressor Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D012319:RNA Polymerase II