Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element is regulated by the essential viral Tat protein that binds to the viral TAR RNA target and recruits a positive transcription elongation complex (P-TEFb). We have used a stepwise transcription approach and a highly sensitive assay to determine the dynamics of interactions between HIV-1 Tat and the transcription complexes actively engaged in elongation. Our results demonstrate that Tat protein associates with RNA polymerase II complexes during early transcription elongation after the promoter clearance and before the synthesis of full-length TAR RNA transcript. This interaction of Tat with RNA polymerase II elongation complexes is P-TEFb-independent. Our results also show that there are two Tat binding sites on each transcription elongation complex; one is located on TAR RNA and the other one on RNA polymerase II near the exit site for nascent mRNA transcripts. These findings suggest that two Tat molecules are involved in performing various functions during a single round of HIV-1 mRNA synthesis.
J Mol Biol 2002 Jul 26
PMID:A bimolecular mechanism of HIV-1 Tat protein interaction with RNA polymerase II transcription elongation complexes. 1212 15

We have partially reconstituted 20S proteasome/RNA complexes using oligonucleotides corresponding to ARE (adenosine- and uridine-rich element) (AUUUA)4 and HIV-TAR (human immunodeficiency virus-Tat transactivation response element), a stem-loop structure in the 5' UTR (untranslated region) of HIV-mRNAs. We demonstrate that these RNAs which associate with proteasomes are degraded by proteasomal endonuclease activity. The formation of these 20S proteasome/RNA substrate complexes is rather specific since 20S proteasomes do not interfere with truncated TAR that is not cleaved by proteasomal endonuclease. In addition, affinity of proteasomes for (AUUUA)4 is much stronger as it is for HIV-TAR. These results provide further arguments for our hypothesis that proteasomes could be involved in the destabilisation of cytokines mRNAs containing AUUUA sequences as well as viral mRNAs.
Mol Biol Rep 2003 Mar
PMID:Substrate affinity and substrate specificity of proteasomes with RNase activity. 1268 29

Nucleic acid-based antiviral approaches have been tried against multiple HIV-1 genes with the purpose of down-regulating its replication. A unique stem-loop structure called TAR is present at the 5'-end of all HIV-1 transcripts; Tat and other cellular proteins bind to TAR and thus govern transcription. Therefore, HIV-1 TAR is an attractive target against which various antiviral approaches could be tried. We screened several DNA enzymes (Dz's) containing the 10-23 catalytic motif and a single Dz containing the 8-17 catalytic motif against the HIV-1 TAR RNA. Dz's directed against the predicted single-stranded bulge regions showed sequence-specific cleavage activities. One of the two Dz's, namely Dz-475, showed moderate cleavage activity in complete absence of Mg(2+). Addition of unrelated sequences at the 5'-end of the HIV-1 TAR RNA rendered it susceptible to four additional Dz-mediated cleavages. Both Dz's (470 and 475) showed significant intracellular reduction of HIV-1 gene expression. Dz-475-treated cells showed significant protection against T-tropic and macrophage-tropic HIV-1 challenge. Dz-475-transfected T-lymphocytes, human PBMCs, or chronically infected cell lines showed marked viral resistance. Unique features of this antiviral strategy with respect to HIV-1 gene inhibition are discussed.
Mol Ther 2003 Jun
PMID:Inhibition of HIV-1 gene expression by novel DNA enzymes targeted to cleave HIV-1 TAR RNA: potential effectiveness against all HIV-1 isolates. 1278 56

The HIV transcriptional activator Tat is acetylated by p300 at a single lysine residue in the TAR RNA binding domain. We have generated monoclonal and polyclonal antibodies specific for the acetylated form of Tat (AcTat). Microinjection of anti-AcTat antibodies inhibited Tat-mediated transactivation in cells. Similarly, the p300 inhibitor Lys-CoA and siRNA specific for p300 suppressed Tat transcriptional activity. Full-length synthetic AcTat bound to TAR RNA with the same affinity as unacetylated Tat, but formation of a Tat-TAR-CyclinT1 ternary complex was completely inhibited in the presence of AcTat. We propose that Tat acetylation may help in dissociating the Tat cofactor CyclinT1 from TAR RNA and serve to transfer Tat onto the elongating RNA polymerase II.
Mol Cell 2003 Jul
PMID:Acetylation of Tat defines a cyclinT1-independent step in HIV transactivation. 1288 2

A primary advantage of lentiviral vectors is their ability to pass through the nuclear envelope into the cell nucleus thereby allowing transduction of nondividing cells. Using HIV-based lentiviral vectors, we delivered an anti-CCR5 ribozyme (CCR5RZ), a nucleolar localizing TAR RNA decoy, or Pol III-expressed siRNA genes into cultured and primary cells. The CCR5RZ is driven by the adenoviral VA1 Pol III promoter, while the human U6 snRNA Pol III-transcribed TAR decoy is embedded in a U16 snoRNA (designated U16TAR), and the siRNAs were expressed from the human U6 Pol III promoter. The transduction efficiencies of these vectors ranged from 96-98% in 293 cells to 15-20% in primary PBMCs. A combination of the CCR5RZ and U16TAR decoy in a single vector backbone gave enhanced protection against HIV-1 challenge in a selective survival assay in both primary T cells and CD34(+)-derived monocytes. The lentiviral vector backbone-expressed siRNAs also showed potent inhibition of p24 expression in PBMCs challenged with HIV-1. Overall our results demonstrate that the lentiviral-based vectors can efficiently deliver single constructs as well as combinations of Pol III therapeutic expression units into primary hematopoietic cells for anti-HIV gene therapy and hold promise for stem or T-cell-based gene therapy for HIV-1 infection.
Mol Ther 2003 Aug
PMID:Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs. 1290 42

The targeting of RNA for the design of novel anti-viral compounds has until now proceeded largely without incorporating direct input from structure-based design methodology, partly because of lack of structural data, and complications arising from substrate flexibility. We propose a paradigm to explain the physical mechanism for ligand-induced refolding of trans-activation response element (TAR RNA) from human immunodeficiency virus 1 (HIV-1). Based upon Poisson-Boltzmann analysis of the TAR structure, as bound by a peptide derived from the transcriptional activator protein, Tat, our hypothesis shows that two specific electrostatic interactions are necessary to stabilise the conformation. This result contradicts the belief that a single argininamide residue is responsible for stabilising the TAR fold, as well as the conventional wisdom that electrostatic interactions with RNA are non-specific or dominated by phosphates. We test this hypothesis by using NMR and computational methods to model the interaction of a series of novel inhibitors of the in vitro RNA-binding activities for a peptide derived from Tat. A subset of inhibitors, including the bis-guanidine compound rbt203 and its analogues, induce a conformation in TAR similar to that brought about by the protein. Comparison of the interactions of two of these ligands with the RNA and structure-activity relationships observed within the compound series, confirm the importance of the two specific electrostatic interactions in the stabilisation of the Tat-bound RNA conformation. This work illustrates how the use of medicinal chemistry and structural analysis can provide a rational basis for prediction of ligand-induced conformational change, a necessary step towards the application of structure-based methods in the design of novel RNA or protein-binding drugs.
J Mol Biol 2004 Feb 13
PMID:Rational design of inhibitors of HIV-1 TAR RNA through the stabilisation of electrostatic "hot spots". 1475 49

The targeting of RNA for the design of novel anti-viral compounds represents an area of vast potential. We have used NMR and computational methods to model the interaction of a series of synthetic inhibitors of the in vitro RNA binding activities of a peptide derived from the transcriptional activator protein, Tat, from human immunodeficiency virus type 1. Inhibition has been measured through the monitering of fluorescence resonance energy transfer between fluorescently labeled peptide and RNA components. A series of compounds containing a bi-aryl heterocycle as one of the three substituents on a benzylic scaffold, induce a novel, inactive TAR conformation by stacking between base-pairs at the site of a three-base bulge within TAR. The development of this series resulted in an enhancement in potency (with Ki < 100 nM in an in vitro assay) and the removal of problematic guanidinium moieties. Ligands from this series can act as inhibitors of Tat-induced transcription in a cell-free system. This study validates the drug design strategy of using a ligand to target the RNA receptor in a non-functional conformation.
J Mol Biol 2004 Feb 20
PMID:Structure-based drug design targeting an inactive RNA conformation: exploiting the flexibility of HIV-1 TAR RNA. 1509 77

The viral nucleic acid chaperone protein NCp7 of HIV-1 assists the two obligatory strand transfers required for the conversion of the genomic RNA into double-stranded DNA by reverse transcriptase. The first strand transfer necessitates the annealing of the early product of cDNA synthesis, the minus strand strong stop DNA (ss-cDNA) to the 3' end of the genomic RNA. The hybridization reaction involves regions containing imperfect stem-loop (SL) structures, namely the TAR RNA at the 3' end of the genomic RNA and the complementary sequence cTAR at the 3' end of ss-cDNA. To pursue the characterization of the interaction between NCp7 and cTAR DNA, we investigated by absorbance, steady-state and time-resolved fluorescence spectroscopy, the interaction of NCp7 with wild-type and mutated DNAs representing the top half of cTAR. NCp7 was found to activate the transient melting of this cTAR DNA structure but less efficiently than that of cTAR lower half. The NCp7-induced destabilization of cTAR top half is dependent upon the three nucleotides bulging out of the stem, which thus represent a melting initiation site. In contrast, despite its ability to bind NCp7, the top loop does not play any significant role in NCp7-mediated melting. Thermodynamic data further suggest that NCp7-mediated destabilization of this cTAR structure correlates with the free energy changes afforded by destabilizing motifs like loops and bulges within the SL secondary structure. Interestingly, since NCp7 melts only short double-stranded sequences, destabilizing motifs need to be regularly positioned along the genomic sequence in order to promote strand transfer and thus genetic recombination during proviral DNA synthesis.
J Mol Biol 2004 May 07
PMID:Role of the structure of the top half of HIV-1 cTAR DNA on the nucleic acid destabilizing activity of the nucleocapsid protein NCp7. 1509 39

The HEXIM1 protein inhibits the kinase activity of P-TEFb (CDK9/cyclin T) to suppress RNA polymerase II transcriptional elongation in a process that specifically requires the 7SK snRNA, which mediates the interaction of HEXIM1 with P-TEFb. In an attempt to define the sequence requirements for HEXIM1 to interact with 7SK and inactivate P-TEFb, we have identified the first 18 amino acids within the previously described nuclear localization signal (NLS) of HEXIM1 as both necessary and sufficient for binding to 7SK in vivo and in vitro. This 7SK-binding motif was essential for HEXIM1's inhibitory action, as the HEXIM1 mutants with this motif replaced with a foreign NLS failed to interact with 7SK and P-TEFb and hence were unable to inactivate P-TEFb. The 7SK-binding motif alone, however, was not sufficient to inhibit P-TEFb. A region C-terminal to this motif was also required for HEXIM1 to associate with P-TEFb and suppress P-TEFb's kinase and transcriptional activities. The 7SK-binding motif in HEXIM1 contains clusters of positively charged residues reminiscent of the arginine-rich RNA-binding motif found in a wide variety of proteins. Part of it is highly homologous to the TAR RNA-binding motif in the human immunodeficiency virus type 1 (HIV-1) Tat protein, which was able to restore the 7SK-binding ability of a HEXIM1 NLS substitution mutant. We propose that a similar RNA-protein recognition mechanism may exist to regulate the formation of both the Tat-TAR-P-TEFb and the HEXIM1-7SK-P-TEFb ternary complexes, which may help convert the inactive HEXIM1/7SK-bound P-TEFb into an active one for Tat-activated and TAR-dependent HIV-1 transcription.
Mol Cell Biol 2004 Jun
PMID:A human immunodeficiency virus type 1 Tat-like arginine-rich RNA-binding domain is essential for HEXIM1 to inhibit RNA polymerase II transcription through 7SK snRNA-mediated inactivation of P-TEFb. 1516 77

We report the design and validation of a fast empirical function for scoring RNA-ligand interactions, and describe its implementation within RiboDock, a virtual screening system for automated flexible docking. Building on well-known protein-ligand scoring function foundations, features were added to describe the interactions of common RNA-binding functional groups that were not handled adequately by conventional terms, to disfavour non-complementary polar contacts, and to control non-specific charged interactions. The results of validation experiments against known structures of RNA-ligand complexes compare favourably with previously reported methods. Binding modes were well predicted in most cases and good discrimination was achieved between native and non-native ligands for each binding site, and between native and non-native binding sites for each ligand. Further evidence of the ability of the method to identify true RNA binders is provided by compound selection ('enrichment factor') experiments based around a series of HIV-1 TAR RNA-binding ligands. Significant enrichment in true binders was achieved amongst high scoring docking hits, even when selection was from a library of structurally related, positively charged molecules. Coupled with a semi-automated cavity detection algorithm for identification of putative ligand binding sites, also described here, the method is suitable for the screening of very large databases of molecules against RNA and RNA-protein interfaces, such as those presented by the bacterial ribosome.
J Comput Aided Mol Des 2004 Mar
PMID:Validation of an empirical RNA-ligand scoring function for fast flexible docking using Ribodock. 1536 19


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