Sp1-dependent activation of a synthetic promoter by human immunodeficiency virus type 1 Tat protein.
The Tat protein coded by human immunodeficiency virus (HIV) is a strong activator of viral gene expression from the long terminal repeat (LTR). It appears that Tat-mediated trans-activation of the HIV LTR is predominantly a transcriptional event. It has been reported that Tat acts at the level of both transcriptional initiation and elongation through interaction with a nascent RNA target sequence termed TAR (for trans-activation response element). However, the precise mechanism(s) by which Tat mediates TAR-dependent transcriptional activity is not known. To determine whether Tat functions similarly to other eukaryotic transcriptional activators through any of the conventional promoter elements, we tested Tat activity on synthetic promoters containing consensus sequences required for binding transcription factor Sp1 and a TATA box. Here, we report that a chimeric Tat protein targeted to the promoter region by the DNA-binding domain of yeast transcription factor GAL4 activates the synthetic promoter. Because this trans-activation depends on Sp1-binding sites, Tat can apparently mediate transcriptional activation through its interaction with Sp1. Mutational analysis of the gal4-tat chimeric gene reveals that the N-terminal 48-amino acid region of Tat constitutes the activation region for Sp1-dependent trans-activation. This region of Tat exhibits substantially more activity than the N-terminal 58 amino acids of Tat, which includes the arginine-rich basic region. Effects of specific mutations in the 48-amino acid Tat region of GAL4-Tat on trans-activation of the synthetic promoter mimic the effects of these specific mutations on Tat-mediated trans-activation of the HIV-1 LTR, suggesting that trans-activation of both the synthetic promoter and the intact LTR occurs by a common mechanism.
Institute for Molecular Virology, St. Louis University Medical Center, MO 63110.
J
J
Kamine
T
T
Subramanian
G
G
Chinnadurai
D001483:Base Sequence; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000502:physiology; D005656:Fungal Proteins / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000502:physiology*; D015497:HIV-1 / Q000235:genetics*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D029701:Saccharomyces cerevisiae Proteins; D016329:Sp1 Transcription Factor / Q000502:physiology*; D014157:Transcription Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D015696:Gene Products, tat; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus