Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have extracted from a random population of about 10(9) oligodeoxynucleotides a series of 21-mers that are able to bind to a folded DNA 76-mer used as a template for in vitro transcription of the TAR element of the retrovirus HIV-1, by the T7 RNA polymerase. Five aptastrucs, that is, aptamers able to bind to the structure, out of 15 analyzed sequences, share the consensus motif 5'-PyGGG(TG)PyC, complementary in part to a weak double-stranded region of the target. (The parentheses indicate that either T or G is missing in one of these aptastrucs.) A dissociation constant of about 3 microM was evaluated by electrophoretic mobility shift assay for the winner sequence. Interactions between the aptastruc and the target sequences involve more than Watson-Crick base pairing of the consensus octamer. The binding is chemistry dependent. Phosphorothioate oligodeoxyribonucleotides and 2'-O-methyl oligoribonucleotides derived from the selected aptastrucs exhibit a weak if any affinity for the target.
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PMID:Identification of aptamers against the DNA template for in vitro transcription of the HIV-1 TAR element. 930 89

Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR. We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors. Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation. The sequence of CA150 contains an extensive glutamine- and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster. Immunodepletion of CA150 abolished Tat trans activation in vitro. Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation. Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II holoenzyme. Furthermore, we found that functional Tat associates with the holoenzyme whereas activation-deficient Tat mutants do not. Thus, we propose that Tat action is transduced via an RNA polymerase II holoenzyme that contains CA150.
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PMID:CA150, a nuclear protein associated with the RNA polymerase II holoenzyme, is involved in Tat-activated human immunodeficiency virus type 1 transcription. 931 62

Artificial recruitment of TATA-binding protein (TBP) to many eukaryotic promoters bypasses DNA-bound activator function. The human immunodeficiency virus type 1 (HIV-1) Tat is an unconventional activator that up-regulates transcription from the HIV-1 long terminal repeat (LTR) through binding to a nascent RNA sequence, TAR. Because this LTR and its cognate activator have atypical features compared to a standard RNA polymerase II (RNAP II) transcriptional unit, the precise limiting steps for HIV-1 transcription and how Tat resolves these limitations remain incompletely understood. We thus constructed human TBP fused to the DNA-binding domain of GAL4 to determine whether recruitment of TBP is one rate-limiting step in HIV-1 LTR transcription and whether Tat functions to recruit TBP. As a control, we compared the activity of the adenovirus E1b promoter. Our findings indicate that TBP tethering to the E1b promoter fully effected transcription to the same degree achievable with the potent GAL4-VP16 activator. By contrast, TBP recruitment to the HIV-1 LTR, although necessary for conferring Tat responsiveness, did not bypass a physical need for Tat in achieving activated transcription. These results document that the HIV-1 and the E1b promoters are transcriptionally limited at different steps; the major rate-limiting step for E1b is recruitment of TBP, while activation of the HIV-1 LTR requires steps in addition to TBP recruitment. We suggest that Tat acts to accelerate rate-limiting steps after TBP recruitment.
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PMID:Promoter activity of Tat at steps subsequent to TATA-binding protein recruitment. 937 21

A strong transcriptional pause delays human RNA polymerase II three nt after the last potentially paired base in HIV-1 TAR, the RNA structure that binds the transactivator protein Tat. We report here that the HIV-1 pause depends in part on an alternative RNA structure (the HIV-1 pause hairpin) that competes with formation of TAR. By probing the nascent RNA structure in halted transcription complexes, we found that the transcript folds as the pause hairpin before and at the pause, and rearranges to TAR concurrent with or just after escape from the pause. The pause signal triggers a 2 nt reverse translocation by RNA polymerase that may block the active site and be counteracted by formation of TAR. Thus, the HIV-1 pause site modulates nascent RNA rearrangement from a structure that favors pausing to one that both recruits Tat and promotes escape from the pause.
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PMID:Transcriptional pausing at +62 of the HIV-1 nascent RNA modulates formation of the TAR RNA structure. 965 86

During transcription of mRNA genes, there is a correlation between the phosphorylation state of the C-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II) and the ability of the RNAP II complex to processively transcribe the gene. To examine the involvement of CTD phosphorylation in modulation of RNAP II function, we have analyzed the ability of a known CTD kinase, human Cdk8, to modulate HIV-1 LTR-driven gene expression upon directed targeting to a promoter-proximal nascent RNA element. The results indicated that Cdk8, when localized to an RNA element, activates gene expression in a catalysis-dependent manner. Also, Cdk8 targeted to RNA was observed to act in a synergystic manner with DNA-targeted Sp1 but not with DNA-targeted HIV-1 Tat, suggesting that RNA-targeted Cdk8 acts on similar rate limiting post-initiation events as Tat. As recent observations suggest that Tat/TAR-mediated transcription of the proviral genome of HIV depends on specific phosphorylation of RNAP II in its CTD by the Tat-associated kinase (TAK/p-TEFb/Cdk9), our results indicate that Cdk8 shares with Cdk9 the ability to modulate transcription upon targeting to a nascent RNA element.
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PMID:Targeting of CDK8 to a promoter-proximal RNA element demonstrates catalysis-dependent activation of gene expression. 968 96

Tat stimulation of HIV-1 transcriptional elongation is species-specific and is believed to require a specific cellular cofactor present in many human and primate cells but not in nonpermissive rodent cells. Human P-TEFb, composed of Cdk9 and cyclin T1, is a general transcription elongation factor that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have also implicated P-TEFb as a Tat-specific cellular cofactor and, in particular, human cyclin T1 as responsible for the species-specific Tat activation. To obtain functional evidence in support of these hypotheses, we generated and examined the activities of human-rodent "hybrid" P-TEFb complexes. We found that P-TEFb complexes containing human cyclin T1 complexed with either human or rodent Cdk9 supported Tat transactivation and interacted with the Tat activation domain and the HIV-1 TAR RNA element to form TAR loop-dependent ribonucleoprotein complexes. Although a stable complex containing rodent cyclin T1 and human Cdk9 was capable of phosphorylating CTD and mediating basal HIV-1 elongation, it failed to interact with Tat and to mediate Tat transactivation, indicating that the abilities of P-TEFb to support basal elongation and Tat activation can be separated. Together, our data indicated that the specific interaction of human P-TEFb with Tat/TAR, mostly through cyclin T1, is crucial for P-TEFb to mediate a Tat-specific and species-restricted activation of HIV-1 transcription. Amino acid residues unique to human Cdk9 also contributed partially to the formation of the P-TEFb-Tat-TAR complex. Moreover, the cyclin box of cyclin T1 and its immediate flanking region are largely responsible for the specific P-TEFb-Tat interaction.
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PMID:Specific interaction of Tat with the human but not rodent P-TEFb complex mediates the species-specific Tat activation of HIV-1 transcription. 1007 79

Tat activates transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by increasing the processivity of RNA polymerase II. Recently, it has been demonstrated that the cellular kinase CDK9 and its binding partner cyclin T1 are involved in regulating transcriptional elongation and tat-activation. Cyclin T1, CDK9 and Tat bind as a complex to elements in TAR RNA that are required for tat-activation. Here, we used cyclin T1 mutants to define domains in this protein that bind to both CDK9 and Tat and are involved in stimulating tat-activation. The region of cyclin T1 extending from amino acid residues 1 to 263 is necessary for complex formation with Tat bound to TAR RNA and for stimulation of tat-activation in murine cells that are normally poorly responsive to the actions of Tat. In contrast, a smaller region of cyclin T1 was required to bind to CDK9 and stimulate its kinase activity. Recombinant cyclin T1 and CDK9 stimulated both basal and tat-induced in vitro transcriptional elongation from the HIV-1 LTR. The effects of Tat on transcriptional elongation may be mediated by its ability to increase CDK9 phosphorylation of the RNA polymerase II C-terminal domain. These results demonstrate that cyclin T1 interactions with Tat and TAR RNA are critical for activation of HIV-1 gene expression.
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PMID:Cyclin T1 domains involved in complex formation with Tat and TAR RNA are critical for tat-activation. 1032 25

Actinomycin D and alpha-amanitin are commonly used to inhibit transcription. Unexpectedly, however, the transcription of the human immunodeficiency virus (HIV-1) long terminal repeats (LTR) is shown to be activated at the level of elongation, in human and murine cells exposed to these drugs, whereas the Rous sarcoma virus LTR, the human cytomegalovirus immediate early gene (CMV), and the HSP70 promoters are repressed. Activation of the HIV LTR is independent of the NFkappaB and TAR sequences and coincides with an enhanced average phosphorylation of the C-terminal domain (CTD) from the largest subunit of RNA polymerase II. Both the HIV-1 LTR activation and the bulk CTD phosphorylation enhancement are prevented by several CTD kinase inhibitors, including 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole. The efficacies of the various compounds to block CTD phosphorylation and transcription in vivo correlate with their capacities to inhibit the CDK9/PITALRE kinase in vitro. Hence, the positive transcription elongation factor, P-TEFb, is likely to contribute to the average CTD phosphorylation in vivo and to the activation of the HIV-1 LTR induced by actinomycin D.
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PMID:The transcriptional inhibitors, actinomycin D and alpha-amanitin, activate the HIV-1 promoter and favor phosphorylation of the RNA polymerase II C-terminal domain. 1034 61

The human immunodeficiency virus type 1 transcriptional regulator Tat increases the efficiency of elongation, and complexes containing the cellular kinase CDK9 have been implicated in this process. CDK9 is part of the Tat-associated kinase TAK and of the elongation factor P-TEFb (positive transcription elongation factor-b), which consists minimally of CDK9 and cyclin T. TAK and P-TEFb are both able to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II, but their relationships to one another and to the stimulation of elongation by Tat are not well characterized. Here we demonstrate that human cyclin T1 (but not cyclin T2) interacts with the activation domain of Tat and is a component of TAK as well as of P-TEFb. Rodent (mouse and Chinese hamster) cyclin T1 is defective in Tat binding and transactivation, but hamster CDK9 interacts with human cyclin T1 to give active TAK in hybrid cells containing human chromosome 12. Although TAK is phosphorylated on both serine and threonine residues, it specifically phosphorylates serine 5 in the CTD heptamer. TAK is found in the nuclear and cytoplasmic fractions of human cells as a large complex (approximately 950 kDa). Magnesium or zinc ions are required for the association of Tat with the kinase. We suggest a model in which Tat first interacts with P-TEFb to form the TAK complex that engages with TAR RNA and the elongating transcription complex, resulting in hyperphosphorylation of the CTD on serine 5 residues.
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PMID:Human and rodent transcription elongation factor P-TEFb: interactions with human immunodeficiency virus type 1 tat and carboxy-terminal domain substrate. 1036 92

The HIV-1-encoded Tat protein controls transcription elongation by increasing processivity of RNA polymerase II (Pol II). Here, we have identified a Tat stimulatory activity (Tat-SF) as a novel RNA Pol II-containing complex. Remarkably, Tat-SF contains the previously identified Tat cofactors Tat-SF1, P-TEFb and hSPT5/Tat-CT1, in addition to RNA Pol II and other unidentified polypeptides, but none of the SRB/MED proteins or other factors found associated with the previously described RNA Pol II holoenzyme complex. Tat-SF supports basal, Sp1-activated and Tat-activated transcription in a reconstituted system, and a Tat-SF-derived fraction lacking RNA Pol II can complement non-responsive RNA Pol II complexes for Tat-enhanced HIV-1 transcription, indicating that Tat-SF contains factors that are critical for Tat function. Both Tat-SF and RNA Pol II holoenzyme are present in HeLa nuclear extracts and each can be recruited to the HIV-1 promoter. Our results indicate that Tat-SF is a Tat cofactor-containing RNA Pol II complex whose recruitment to the promoter provides elongation factors important for Tat-enhanced HIV-1 transcription following TAR RNA synthesis.
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PMID:A novel RNA polymerase II-containing complex potentiates Tat-enhanced HIV-1 transcription. 1039 84


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