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

It has been suggested that ligand-dependent gene activation by the progesterone receptor (PR) can result from recruitment of PR by the promoter bound Sp1. A detailed investigation of the Sp1-dependent agonistic activity of RU486 and R5020 on the folate receptor (FR) type alpha, p27, thymidine kinase 1 and p21 genes reveals a different mechanism. The FR-alpha P4 promoter and the endogenous FR-alpha gene were up-regulated by the PR agonist R5020 through either PR-A or PR-B. The classical antagonist RU486 also activated the promoter but only through PR-B. The most proximal (essential) G/C-rich (Sp1 binding) element and the initiator region constituted the minimal promoter responsive to PR regulation; substitution with a stronger cluster of G/C-rich elements enhanced the magnitude of the PR response. In contrast, substitution of the G/C-rich element with a TATA box resulted in the loss of regulation by PR. Overexpression of Sp1 and Sp4 but not Sp3 enhanced activation of the FR-alpha promoter by PR, knocking down Sp1 decreased the activation in a manner that was reversed by ectopic Sp1 or Sp4. The ligand-dependent action of PR on the promoter was delayed compared with its activation of a classical glucocorticoid response element-driven promoter and activation of both the promoter and the endogenous FR-alpha gene by PR required new protein synthesis. Activation by PR paralleled RNA polymerase II recruitment but was not accompanied by either association of PR or a change in the association of Sp1 with the endogenous FR-alpha P4 promoter. Similar observations were made for PR regulation of the genes encoding p27, thymidine kinase 1, and p21. The results contradict the current view of Sp1-dependent gene regulation by PR and point to the existence of one or more PR target genes whose promoter and cell context(s) must thus be key determinants of the agonistic activity of RU486 on a large group of important Sp1-dependent downstream target genes.
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PMID:R5020 and RU486 act as progesterone receptor agonists to enhance Sp1/Sp4-dependent gene transcription by an indirect mechanism. 1719 5

Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.
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PMID:Lethal mutagenesis of poliovirus mediated by a mutagenic pyrimidine analogue. 1768 44

One of the characteristic features of herpesviruses is that most of their genes are intronless. Thus, their replication relies on the selective nuclear export of intronless viral mRNAs, which have to compete with the nuclear export of bulk spliced cellular mRNAs. Therefore, the regulation of nuclear mRNA export is crucial for the replication and pathogenesis of herpesviruses. Besides the thymidine kinase transcript of herpes simplex virus type 1, which contains specific sequences to facilitate the nuclear export of intronless mRNA, other cis-acting RNA elements for nuclear mRNA export have not yet been identified in the rest of herpesviral intronless mRNAs. Instead, emerging studies show that herpesviruses encode viral mRNA export factors, which interact with components of the major cellular mRNA export pathway, the RNA polymerase II transcription complex and specific splicing factors to selectively export intronless herpesviral mRNAs to the cytoplasm in infected cells. These herpesviral mRNA export factors are members of a conserved gene family that can be found in all herpesviruses. The human cytomegalovirus transactivator protein UL69, which has been demonstrated to belong to this conserved protein family, shares common features with its herpesviral homologues but also possesses unique properties that will be discussed in this review.
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PMID:The human cytomegalovirus regulatory protein UL69 and its effect on mRNA export. 1798 67

The binding of herpes simplex virus type 1 ICP4, TATA-binding protein (TBP), and RNA polymerase II (polII) to the promoter regions of representative immediate-early (IE) (ICP0), early (E) (thymidine kinase [tk]), and late (L) (glycoprotein C [gC]) genes on the viral genome was examined as a function of time postinfection, viral DNA replication, cis-acting sites for TFIID in the tk and gC promoters, and genetic background of ICP4. The binding of TBP and polII to the IE ICP0 promoter was independent of the presence of ICP4, whereas the binding of TBP and polII to the tk and gC promoters occurred only when ICP4 also bound to the promoters, suggesting that the presence of ICP4 at the promoters of E and L genes in virus-infected cells is crucial for the formation of transcription complexes on these promoters. When the TATA box of the tk promoter or the initiator element (INR) of the gC promoter was mutated, a reduction in the amount of TBP and polII binding was observed. However, a reduction in the amount of ICP4 binding to the promoters was also observed, suggesting that the binding of TBP-containing complexes and ICP4 is cooperative. The binding of ICP4, TBP, and polII was also observed on the gC promoter at early times postinfection or when DNA synthesis was inhibited, suggesting that transcription complexes may be formed early on L promoters and that additional events or proteins are required for expression. The ability to form these early complexes on the gC promoter required the DNA-binding domain but in addition required the carboxyl-terminal 524 amino acids of ICP4, which is missing the virus n208. This region was not required to form TBP- and polII-containing complexes on the tk promoter. n208 activates E but not L genes during viral infection. These data suggest that a region of ICP4 may differentiate between forming TBP- and polII-containing complexes on E and L promoters.
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PMID:Binding of ICP4, TATA-binding protein, and RNA polymerase II to herpes simplex virus type 1 immediate-early, early, and late promoters in virus-infected cells. 1809 62

Our laboratory has previously identified an important intragenic region in the human immunodeficiency virus type 1 (HIV-1) genome, whose complete functional unit is composed of the 5103 fragment, the DNaseI-hypersensitive site HS7 and the 5105 fragment. These fragments (5103 and 5105) both exhibit a phorbol 12-myristate 13-acetate (PMA)-inducible enhancer activity on the herpes simplex virus thymidine kinase promoter. Here, we characterized the three previously identified AP-1 binding sites of fragment 5103 by showing the PMA-inducible in vitro binding and in vivo recruitment of c-Fos, JunB and JunD to this fragment located at the end of the pol gene. Functional analyses demonstrated that the intragenic AP-1 binding sites are fully responsible for the PMA-dependent enhancer activity of fragment 5103. Moreover, infection of T-lymphoid Jurkat and promonocytic U937 cells with wild-type and mutant viruses demonstrated that mutations of the intragenic AP-1 sites individually or in combination altered HIV-1 replication. Importantly, mutations of the three intragenic AP-1 sites led to a decreased in vivo recruitment of RNA polymerase II to the viral promoter, strongly supporting that the deleterious effect of these mutations on viral replication occurs, at least partly, at the transcriptional level. Single-round infections of monocyte-derived macrophages confirmed the importance of intragenic AP-1 sites for HIV-1 infectivity.
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PMID:The AP-1 binding sites located in the pol gene intragenic regulatory region of HIV-1 are important for viral replication. 2152 60

ICP4 is the major activator of herpes simplex virus (HSV) transcription. Previous studies have defined several regions of ICP4 that are important for viral gene expression, including a DNA binding domain and transactivation domains that are contained in the C-terminal and N-terminal 520 and 274 amino acids, respectively. Here we show that the N-terminal 210 amino acids of ICP4 are required for interactions with components of TFIID and mediator and, as a consequence, are necessary for the activation of viral genes. A mutant of ICP4 deleted for amino acids 30 to 210, d3-10, was unable to complement an ICP4 null virus at the level of viral replication. This was the result of a severe deficiency in viral gene and protein expression. The absence of viral gene expression coincided with a defect in the recruitment of RNA polymerase II to a representative early promoter (thymidine kinase [TK]). Affinity purification experiments demonstrated that d3-10 ICP4 was not found in complexes with components of TFIID and mediator, suggesting that the defect in RNA polymerase II (Pol II) recruitment was the result of ablated interactions between d3-10 and TFIID and mediator. Complementation assays suggested that the N-terminal and C-terminal regions of ICP4 cooperate to mediate gene expression. The complementation was the result of the formation of more functional heterodimers, which restored the ability of the d3-10-containing molecules to interact with TFIID. Together, these studies suggest that the N terminus contains a true activation domain, mediating interactions with TFIID, mediator, and perhaps other transcription factors, and that the C terminus of the molecule contains activities that augment the functions of the activation domain.
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PMID:Requirement of the N-terminal activation domain of herpes simplex virus ICP4 for viral gene expression. 2313 15


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