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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcriptional termination by RNA polymerase II at the 3' end of genes encoding poly(A)+ mRNAs is thought to require two distinct cis-active elements: a functional poly(A) signal and a downstream transcriptional pause site. An important requirement for efficient termination is to prevent transcriptional interference of downstream-located promoters. We have therefore investigated whether these two elements, individually or in combination, can prevent transcriptional interference of RNA polymerase II-activated promoters. For this purpose, we constructed an expression plasmid containing two tandem retroviral long terminal repeats (LTRs) derived from HIV-1. When transfected into HeLa cells, this construct resulted in transcriptional interference of the LTR promoters. Using this assay, we were able to show that a single poly(A) signal was able to protect an otherwise occluded promoter. This effect depended on the RNA-processing strength of the poly(A) signal. Furthermore, transcriptional pause sites provided adequate protection against promoter occlusion even when tested alone. Finally, a combined element consisting of a poly(A) signal followed by a pause site was more efficient in promoter protection than either element on its own. These results indicate that an interference-blocking element can take various forms: a poly(A) signal, a transcriptional pause site or a combination of both.
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PMID:Poly(A) signals and transcriptional pause sites combine to prevent interference between RNA polymerase II promoters. 850 77

NF-kappa B is a potent inducible transcription factor that regulates many genes in activated T cells. In this report we examined the ability of different subunits of NF-kappa B to enhance HIV-1 transcription in vitro with chromatin templates. We find that the p65 subunit of NF-kappa B is a strong transcriptional activator of nucleosome-assembled HIV-1 DNA, whereas p50 does not activate transcription, and that p65 activates transcription synergistically with Sp1 and distal HIV-1 enhancer-binding factors (LEF-1, Ets-1, and TFE-3). These effects were observed with chromatin, but not with nonchromatin templates. Furthermore, binding of either p50 or p65 with Sp1 induces rearrangement of the chromatin to a structure that resembles the one reported previously for integrated HIV-1 proviral DNA in vivo. These results suggest that p50 and Sp1 contribute to the establishment of the nucleosomal arrangement of the uninduced provirus in resting T cells, and that p65 activates transcription by recruitment of the RNA polymerase II transcriptional machinery to the chromatin-repressed basal promoter.
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PMID:NF-kappa B-mediated chromatin reconfiguration and transcriptional activation of the HIV-1 enhancer in vitro. 855 93

A double-stranded RNA structure transcribed from the HIV-1 long terminal repeat known as TAR is critical for increasing gene expression in response to the transactivator protein Tat. Two cellular factors, RNA polymerase II and TRP-185, bind specifically to TAR RNA, but require the presence of cellular proteins known as cofactors which by themselves are unable to bind to TAR RNA. In an attempt to determine the mechanism by which these cofactors stimulate binding to TAR RNA, we purified these factors from HeLa nuclear extract and amino acid microsequence analysis performed. Three proteins were identified in the cofactor fraction including two previously described proteins, elongation factor 1alpha (EF-1alpha) and the polypyrimidine tract-binding protein (PTB), and a novel protein designated the stimulator of TAR RNA-binding proteins (SRB). SRB has a high degree of homology with a variety of cellular proteins known as chaperonins. Recombinant EF-1alpha, PTB, and SRB produced from vaccinia expression vectors stimulated the binding of RNA polymerase II and TRP-185 to TAR RNA in gel retardation analysis. These studies define a group of cellular factors that function in concert to stimulate the binding of TRP-185 and RNA polymerase II to HIV-1 TAR RNA.
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PMID:Identification of a group of cellular cofactors that stimulate the binding of RNA polymerase II and TRP-185 to human immunodeficiency virus 1 TAR RNA. 862 63

A 16-base pair oligo(purine)-oligo(pyrimidine) sequence present in the coding region of two HIV 1 proviral genes (pol and nef) was chosen as a target for triplex-forming oligonucleotides in in vitro transcription assays. Inhibition of transcription elongation was observed with triplex-forming oligonucleotide-acridine conjugates (Acr-15-TCG:5'-Acr-T4CT4G6-3' and Acr-9-TC:5'-Acr-T4CT4-3' where C is 5-methylcytosine) under conditions where the unsubstituted oligomers did not exhibit any inhibitory effect. Both SP6 bacteriophage RNA polymerase and eukaryotic RNA polymerase II were physically blocked by such a triplex barrier. The polymerase arrest is caused by the triple-helical complex involving the hydrogen-bonded oligonucleotide stabilized by the intercalated moiety and not solely by the acridine molecule specifically intercalated at the duplex-triplex junction. The stability of the triple-helical complex formed by the 15-mer containing thymines, cytosine, and guanines (15-TCG) and involving the formation of six contiguous C.GxG base triplets was strongly enhanced in the presence of a benzopyridoindole derivative (BePI), which intercalates in triplex structures. This improvement of the binding affinity led to an increased inhibition of transcription elongation. The present results demonstrate the necessity to use triplex-forming oligonucleotides with high binding affinity and a long residence time on their double-stranded target to efficiently inhibit transcription elongation. These data provide a rational basis for the optimization and the development of triplex-forming oligonucleotides as transcriptional blockers, even when they are targeted to the transcribed portion of a gene, downstream of the transcription initiation site.
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PMID:Specific inhibition of in vitro transcription elongation by triplex-forming oligonucleotide-intercalator conjugates targeted to HIV proviral DNA. 875 10

Activation of HIV-1 gene expression by the transactivator Tat is dependent on an RNA regulatory element located downstream of the transcription initiation site known as TAR. To characterize cellular factors that bind to TAR RNA and are involved in the regulation of HIV-1 transcription, HeLa nuclear extract was fractionated and RNA gel-retardation analysis was performed. This analysis indicated that only two cellular factors, RNA polymerase II and the previously characterized TAR RNA loop binding protein TRP-185, were capable of binding specifically to TAR RNA. To elucidate the function of TRP-185, it was purified from HeLa nuclear extract, amino acid microsequence analysis was performed and a cDNA encoding TRP-185 was isolated. TRP-185 is a novel protein of 1621 amino acids which contains a leucine zipper and potentially a novel RNA binding motif. In gel-retardation assays, the binding of both recombinant TRP-185 and RNA polymerase II was dependent on the presence of an additional group of proteins designated cellular cofactors. Both the TAR RNA loop and bulge sequences were critical for RNA polymerase II binding, while TRP-185 binding was dependent only on TAR RNA loop sequences. Since binding of TRP-185 and RNA polymerase II to TAR RNA was found to be mutually exclusive, our results suggest that TRP-185 may function either alone or in conjunction with Tat to disengage RNA polymerase II which is stalled upon binding to nascently synthesized TAR RNA during transcriptional elongation.
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PMID:The cellular factor TRP-185 regulates RNA polymerase II binding to HIV-1 TAR RNA. 884 92

Adeno-associated virus (AAV) Rep78 is a multifunctional protein that is required for AAV transcriptional activity, AAV DNA replication, and possibly for site-specific integration of AAV into human chromosome 19. Rep78 is also able to inhibit a variety of heterologous promoters, including those of c-H-ras, human papillomavirus types 16 and 18, and HIV type 1. However, Rep78 is unable to significantly affect murine osteosarcomavirus (MSV). It was noticed that promoters that are inhibited possess binding motifs for the cellular transcription factor Sp1, whereas the MSV long terminal repeat promoter did not. These data stimulated the hypothesis that Rep78 may recognize and interact with cellular Sp1. Here, we demonstrate that Rep78 is able to interact with Sp1 in vitro as analyzed by West(far)-Western, electrophoretic mobility shift assay-supershift, and coimmunoprecipitation analyses. Furthermore, in support of an in vivo biological effect from this interaction, Rep78 is demonstrated to inhibit a synthetic, Sp1-dependent promoter. Further still, the insertion of Sp1 DNA binding motifs into the Rep78-resistant MSV long terminal repeat results in a promoter that has increased sensitivity to inhibition by Rep78. Finally, it is demonstrated that the Sp1-Rep78 interaction requires the amino half of Rep78. The interaction of Rep78 with Sp1, along with possible downstream effects on the transcription initiation process of RNA polymerase II, may partially explain the rather broad-based antitumor abilities of AAV.
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PMID:The adeno-associated virus Rep78 major regulatory/transformation suppressor protein binds cellular Sp1 in vitro and evidence of a biological effect. 891 72

The protein Tat is encoded by the HIV-1 genome and is essential for viral replication because of its activation of viral transcription. Tat enhances the ability of RNA polymerase II (Pol II) to move long distances down the DNA through a poorly understood mechanism that involves its binding the to the 5' end of the nascent HIV-1 transcript. It has been suggested that the stimulation of transcript elongation by conventional DNA-binding activators may involve phosphorylation of the carboxy-terminal domain (CTD) of Pol II by the transcription factor TFIIH through the associated CAK kinase. Here we show that Tat-enhanced HIV-1 transcription in vitro requires both TFIIH and the CTD of Pol II. In addition, Tat, through its activation domain, both interacts with a functional TFIIH-containing complex and stimulates phosphorylation of a CTD-containing substrate by the TFIIH kinase. Under conditions that jointly restrict transcriptional elongation and TFIIH-mediated CTD phosphorylation, Tat stimulates both these activities. Furthermore, RNA synthesis is required for Tat to stimulate phosphorylation of the CTD when it is part of an initiation complex, as expected from Tat's interaction with viral transcripts. Thus, stimulation of Pol II elongation by Tat may involve direct effects on TFIIH-mediated CTD phosphorylation.
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PMID:Enhanced processivity of RNA polymerase II triggered by Tat-induced phosphorylation of its carboxy-terminal domain. 893 26

The HIV-1 (human immunodeficiency virus type 1) and HIV-2 Tat proteins increase the level of transcription from their corresponding long terminal repeats. Tat activates transcription likely by interaction with components of the transcriptional initiation and elongation complexes during different stages of the transcription reaction. In the current study, two approaches were used to address the sites at which Tat becomes stably associated with the HIV transcription complex. First, we isolated column purified HIV-1 and HIV-2 transcription complexes that were competent for in vitro transcription and found that wild-type but not mutant Tat protein was specifically associated with this complex. An intact HIV TATA element and the presence of functional TATA-binding protein were necessary for Tat association. In contrast, the HIV-1 and HIV-2 TAR bulge sequences which serve as binding sites for Tat were not required for its association with the HIV preinitiation complex. A second complementary approach using immobilized HIV-1 and HIV-2 templates also demonstrated a functional association of Tat with HIV-1 and HIV-2 preinitiation complexes. Wild-type but not mutant Tat proteins associated with transcription complexes assembled on immobilized HIV-1 and HIV-2 templates and the association of Tat correlated with increases in the level of in vitro transcription. These results indicate that Tat can associate with HIV-1 and HIV-2 transcription complexes prior to the initiation of transcription by RNA polymerase II.
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PMID:Association of Tat with purified HIV-1 and HIV-2 transcription preinitiation complexes. 905 83

The Tat protein is a transcriptional activator which is required for efficient human immunodeficiency virus 1 (HIV-1) gene expression Tat stimulates HIV-1 transcriptional elongation by increasing the processivity of RNA polymerase II. To address whether Tat-mediated effects on HIV-1 gene expression are due to modulation in the phosphorylation of the RNA polymerase II C-terminal domain (CTD), we developed a purification protocol to identify cellular kinases that are capable of binding to Tat and hyperphosphorylating the RNA polymerase II CTD. A 600 kDa protein complex with these properties was isolated, and specific components were identified using peptide microsequence analysis. This analysis indicated that proteins comprising the multi-subunit TFIIH complex, in addition to several novel factors, were associated with Tat using both in vitro and in vivo analysis. The Tat-associated kinase bound to the activation domain of Tat, and its ability to hyperphosphorylate RNA polymerase II was markedly stimulated by Tat. Furthermore, the addition of the Tat-associated kinase to in vitro transcription assays stimulated the ability of Tat to activate HIV-1 transcription. These results define a cellular kinase complex whose activity is modulated by Tat to result in activation of HIV-1 trancription.
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PMID:Purification of a Tat-associated kinase reveals a TFIIH complex that modulates HIV-1 transcription. 918 28

We have developed a new biochemical method to isolate a homogeneous population of RNA polymerase II (RNA pol II) elongation complexes arrested at a DNA damage site. The method involves triple-helix formation at a predetermined site in DNA template with a third strand labeled with psoralen at its 5'-end and a biotin at the 3'-end. After triplex formation and near-ultraviolet irradiation (360 nm), DNA templates modified with psoralen were immobilized on streptavidin-coated magnetic beads and used for in vitro transcription reactions with HeLa nuclear extracts. Separation of magnetic beads from solution results in isolation of arrested elongation complexes on the immobilized DNA templates. We have applied the method to arrest RNA pol II elongation complexes on a DNA template containing HIV-1 promoter. Our results indicate that psoralen crosslink in the template strand efficiently arrests elongation complexes, and psoralen monoadducts terminate transcription. Our results also demonstrate that a triple-helical structure stabilized by an intercalator, acridine, attached to the third strand of the helix inhibits transcription by a termination pathway. Isolation of stable RNA pol II elongation complexes arrested at DNA damage sites is a remarkable finding. This result demonstrates that arrested elongation complexes are impervious to DNA damage repair machinery and other regulatory proteins present in HeLa nuclear extracts. The method of delivering site-specific psoralen damage by a triplex structure and isolation of arrested RNA pol II elongation complexes should be generalizable to any promoter and DNA template sequences. This strategy provides a new approach to study the mechanism of transcription elongation and transcription-coupled DNA damage repair.
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PMID:DNA damage-dependent transcriptional arrest and termination of RNA polymerase II elongation complexes in DNA template containing HIV-1 promoter. 919 26


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