Greenberg, M. E., Mathews, M. B. (December 1997) Effects of heterologous downstream sequences on the activity of the HIV-1 promoter and its response to Tat. Nucleic Acids Research, 25 (24). pp. 5017-24. ISSN 0305-1048
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Abstract
In HIV-1 infection, Tat acts at least in part to control transcriptional elongation by overcoming premature transcriptional termination. In some other genes this process is governed by DNA elements called attenuators in concert with cellular transcription factors. To understand the action of Tat more fully and explore its role as an anti-attenuator, we examined the ability of several natural and synthetic attenuation sequences to modulate transcription initiated at the HIV LTR. Fragments containing these signals were inserted downstream of the TAR element in an HIV-CAT chimera and their effects on transcription were assessed both in vitro and in vivo. Runoff transcription assays in HeLa cell extracts demonstrated that the attenuators give rise to premature termination of transcripts initiated from the heterologous HIV-LTR promoter in vitro. When transiently expressed following transfection into Cos cells, however, premature transcript termination at the attenuation site was not observed. Nevertheless, many of the inserted sequences exerted marked effects on CAT gene expression and on transactivation by Tat at both the RNA and protein levels. The nature and magnitude of the effects depended upon the identity of the attenuator and its orientation but only one of 16 sequences tested met the criteria for a Tat-suppressible attenuator in vivo. One other sequence, in contrast, severely reduced Tat-activated transcription without inhibiting basal transcription These results indicate that sequences downstream of the HIV LTR can influence its function as a promoter and its response to Tat transactivation, but lend little support to their role as attenuators in vivo.
| Item Type: | Paper |
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| Uncontrolled Keywords: | Animals COS Cells Chloramphenicol O-Acetyltransferase/metabolism Gene Expression Regulation, Viral Gene Products, tat/ genetics Genes, Synthetic Genes, fos Genes, myc HIV-1/ genetics Hela Cells Humans Ornithine Decarboxylase/metabolism Recombinant Fusion Proteins/biosynthesis/genetics Regulatory Sequences, Nucleic Acid Repetitive Sequences, Nucleic Acid/ genetics Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Transcription, Genetic |
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > transcription diseases & disorders > viral diseases > HIV bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene expression |
| CSHL Authors: | |
| Communities: | CSHL labs > Skowronski lab |
| Depositing User: | Kathleen Darby |
| Date: | 15 December 1997 |
| Date Deposited: | 07 May 2014 15:07 |
| Last Modified: | 07 May 2014 15:07 |
| PMCID: | PMC147141 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/30026 |
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