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At least 937 records · Page 52Linked to original sources
PubMed2003

Solid-phase synthesis of diamine and polyamine amino acid derivatives as HIV-1 tat-TAR binding inhibitors.

A series of diamine and polyamine derivatives, either free amines or salts (HCl or TFA), of aspartic and glutamic acid were prepared in excellent yields using Rink Amide solid-phase synthesis. The asparagine and glutamine derivatives were all evaluated for their ability to inhibit Tat-TAR binding using a FIGS cellular assay, with the polyamine derivatives exhibiting the most promising binding activity.

Anti-HIV Agents↗
PubMed1997

Targeting the Tat-binding site of bovine immunodeficiency virus TAR RNA with a shape-selective rhodium complex.

The Tat-binding site of the bovine immunodeficiency virus TAR RNA hairpin has been targeted by Rh(phen)2phi3+ (phen = phenanthroline, phi = 9,10-phenanthrenequinone diimine), a photochemical probe of RNA tertiary structure. The primary site cleaved by the rhodium complex, upon photoactivation, is U24, a base which participates in the novel base triple (with bases A13 and U10) characteristic of this folded RNA. delta-Rh(phen)2phi3+ binds to this site with an affinity of 2 x 10(6) M-1. Upon mutation of U24 and A13 to A24 and U13, respectively, so that the RNA oligomer is unable to form the base triple, site-specific cleavage by the rhodium complex is abolished. Moreover, as determined through rhodium photocleavage, at a concentration of 20 microM, Rh(phen)2phi3+ inhibits specific binding of BIV-Tat peptide (2 microM) to its target site. Thus the rhodium complex, in matching its shape to the opened major groove of the properly folded RNA, specifically targets its site and is able to compete for its target with the BIV-Tat peptide.

Animals↗
PubMed1996

How Tat targets TAR: structure of the BIV peptide-RNA complex.

Two new NMR structures describing the complex formed by a peptide from the BIV Tat protein with the TAR RNA provide a significant advance in our understanding of the ways in which peptides interact with specific sites in the major groove of their RNA targets.

Amino Acid Sequence↗
PubMed1996

New features in RNA recognition: a Tat-TAR complex.

The structure of a peptide from the BIV Tat protein bound to its cognate TAR RNA has been solved. This structure reveals a beta-hairpin motif for the protein, bound at a widened bulge site in the major groove of the RNA.

Gene Products, tat↗
PubMed1996

A docking and modelling strategy for peptide-RNA complexes: applications to BIV Tat-TAR and HIV Rev-RBE.

BACKGROUND: In spite of the great interest in the interaction between RNAs and proteins, no general protocol for modelling these complexes is presently available. This methodological vacuum is particularly acute because the structure of few such complexes is known. RESULTS: A general strategy for docking and modelling RNA-protein complexes has been developed. The docking procedure involves minimizing electrostatic and van der Waals' interaction energies of conformationally rigid structures during docking. After docking, libraries of amino acid sidechain conformations are searched to obtain the best interactions between the peptide and the RNA. Using this method, we have reproduced the structure of a bovine immunodeficiency virus (BIV) Tat peptide bound to BIV TAR RNA and have developed a model for the structure of the arginine-rich HIV-1 Rev peptide (Rev34-50) interacting with the Rev-binding element (RBE). CONCLUSIONS: The resulting model of the Rev34-50-RBE complex predicts that although no single arginine sidechain is responsible for complex formation, residues Arg2, Arg5 and Arg11 are more important for binding than the other arginine residues in the peptide. One model is supported by binding measurements performed on wild-type and mutant RBE molecules with the peptide.

Animals↗
PubMedDate not supplied

A new strategy for site-specific protein modification: analysis of a Tat peptide-TAR RNA interaction.

Site-specific modification of proteins and peptides with reporter molecules provides a powerful research tool in chemistry and biology. We report the synthesis and application of a tyrosine analogue, N-alpha-Fmoc-3-acetyl-L-tyrosine, for selective modification of proteins. As a model system, we synthesized the human immunodeficiency virus type 1 (HIV-1) Tat peptide (amino acids 47-56) containing the arginine rich RNA-binding region and replaced the Tyr-47 with 3-acetyl-tyrosine. The acetyl-Tyr-Tat peptide was subsequently labeled with a fluorescein derivative to study RNA-protein interactions by fluorescence energy transfer experiments. Our results showed that the Tat peptide binds to the rhodamine labeled TAR RNA with a dissociation constant (KD) of 1.0 +/- 0.5 nM. This strategy of selective protein modification offers a versatile new procedure for labeling peptides of biological interest at a desired site when several nucleophilic side chains of lysine and cysteine are present. These methods would provide tools for postsynthetic peptide modification and introducing biophysical probes for structural and functional analysis of proteins.

Contrast Media↗
PubMedDate not supplied

Dual-specificity interaction of HIV-1 TAR RNA with Tat peptide-oligonucleotide conjugates.

An oligonucleotide-peptide conjugate, having dual binding capability for a designated RNA, was designed. The peptide portion of the conjugate interacts with a folded domain in the RNA, whereas the oligonucleotide portion hybridizes with a nearby single-stranded region in the RNA. The dual specificity was proven in a model HIV-1 TAR RNA system using an RNase H cleavage assay to assess antisense binding to this RNA. The peptide portion of the conjugate was shown to confer increased specificity on the oligonucleotide.

Amino Acid Sequence↗
PubMed1997

Structural RNA mimetics: N3'-->P5' phosphoramidate DNA analogs of HIV-1 RRE and TAR RNA form A-type helices that bind specifically to Rev and Tat-related peptides.

An attractive strategy for the development of anti-retroviral drugs is the exploration of compounds that mimic RNA control regions of the viral genome and act as "decoys" to sequester viral gene regulatory proteins. Decoys consisting of RNA, however, are chemically unstable and readily degraded by cellular nucleases. DNA decoys, which are slightly more stable, also might not be appropriate because of possible structural differences between RNA and DNA helices and the complexes they form with proteins. It was recently reported, however, that DNA analogs with modified N3'-->P5' phosphoramidate sugar-phosphate backbones are stable and nuclease-resistant and exist predominately as A-form helices in solution [Gryaznov, S., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5798-5802]. We now report that oligonucleotide N3'-->P5' phosphoramidates DNA analogs of HIV-1 RRE IIB and TAR RNA form stable duplexes that exist in the A form as judged by circular dichroism (CD). Moreover, gel shift assays demonstrate that these phosphoramidates can specifically bind to peptides derived from HIV-1 Rev and Tat proteins. Isosequential phosphodiester DNA duplexes, existing in the B form by CD, do not bind to the respective peptides under the experimental conditions used. These results suggest the possibility that nuclease-resistant oligonucleotide N3'-->P5' phosphoramidates might serve as RNA-like decoys and disrupt specific viral RNA/protein interactions such as RRE/Rev and TAR/Tat in HIV-1.

Binding Sites↗
PubMed2004

Combined application of stable carbon isotope analysis and specific metabolites determination for assessing in situ degradation of aromatic hydrocarbons in a tar oil-contaminated aquifer.

To evaluate the intrinsic bioremediation potential in an anoxic tar oil-contaminated aquifer at a former gasworks site, groundwater samples were qualitatively and quantitatively analyzed by compound-specific isotope analysis (CSIA) and signature metabolites analysis (SMA). 13C/12C fractionation data revealed conclusive evidence for in situ biodegradation of benzene, toluene, o-xylene, m/p-xylene, naphthalene, and 1-methylnaphthalene. In laboratory growth studies, 13C/12C isotope enrichment factors for anaerobic degradation of naphthalene (epsilon = -1.1 +/- 0.4) and 2-methylnaphthalene (epsilon = -0.9 +/- 0.1) were determined with the sulfate-reducing enrichment culture N47, which was isolated from the investigated test site. On the basis of these and other laboratory-derived enrichment factors from the literature, in situ biodegradation could be quantified for toluene, o-xylene, m/p-xylene, and naphthalene. Stable carbon isotope fractionation in the field was also observed for ethylbenzene, 2-methylnaphthalene, and benzothiophene but without providing conclusive results. Further evidence for the in situ turnover of individual BTEX compounds was provided by the presence of acetophenone, o-toluic acid, and p-toluic acid, three intermediates in the anaerobic degradation of ethylbenzene, o-xylene, and p-xylene, respectively. A number of groundwater samples also contained naphthyl-2-methylsuccinic acid, a metabolite that is highly specific for the anaerobic degradation of 2-methylnaphthalene. Additional metabolites that provided evidence on the anaerobic in situ degradation of naphthalenes were 1-naphthoic acid, 2-naphthoic acid, 1,2,3,4-tetrahydronaphthoic acid, and 5,6,7,8-tetrahydronaphthoic acid. 2-Carboxybenzothiophene, 5-carboxybenzothiophene, a putative further carboxybenzothiophene isomer, and the reduced derivative dihydrocarboxybenzothiophene indicated the anaerobic conversion of the heterocyclic aromatic hydrocarbon benzothiophene. The combined application of CSIA and SMA, as two reliable and independent tools to collect direct evidence on intrinsic bioremediation, leads to a substantially improved evaluation of natural attenuation in situ.

Bacteria, Anaerobic↗
PubMed2004

A peptide nucleic acid-neamine conjugate that targets and cleaves HIV-1 TAR RNA inhibits viral replication.

The neamine part of the aminoglycoside antibiotic neomycin B was conjugated to a 16 mer peptide nucleic acid (PNA) targeting HIV-1 TAR RNA. Attachment of the neamine core allows cellular uptake of the PNA and results in potent inhibition of HIV-1 replication. The polycationic neamine moiety imparts greater solubility to the PNA and also confers a unique RNA cleavage property to the conjugate which is specific to its target site and functional at physiological concentrations of Mg(2+). These properties suggest a potential therapeutic application for this class of compounds.

Aminoglycosides↗
PubMed2000

The aspartate chemoreceptor Tar is effectively methylated by binding to the methyltransferase mainly through hydrophobic interaction.

In the chemotaxis of Escherichia coli, adaptation requires the methylation and demethylation of transmembrane receptors, which are catalysed by the methyltransferase CheR and the methylesterase CheB respectively. CheR binds to major chemoreceptors through their C-terminal motif NWETF, which is distinct from the methylation sites. In this study, we carried out a systematic mutagenesis of the pentapeptide sequence of Tar. Receptor methylation and adaptation were severely impaired by the alanine substitution of residue W550 and, to a lesser extent, by that of F553. Substitution of residues N549, E551 and T552 had only a slight or little effect. The defects of the W550A and F553A mutations were suppressed by high- and low-level overproduction of CheR respectively. Expression of a fusion protein containing the NWETF sequence, but not its W550A and F553A versions, inhibited chemotaxis of the Che+ strain. In an in vitro assay, CheR bound to the wild-type version but not to the mutant versions. These results and further mutagenesis suggest that the hydrophobicity and the size of residues W550 and F553 are critical in the interaction with CheR, a conclusion that is consistent with the crystal structure of a CheR-NWETF complex. On the other hand, the negatively charged side chain of E551 and the polar side chains of N549 and T552 may not be strictly required, although the presence of a salt bridge and hydrogen bonds between these residues and residues from CheR has been noted in the co-crystal.

Aspartic Acid↗
PubMed2005

Carbon starvation in glacial trees recovered from the La Brea tar pits, southern California.

The Rancho La Brea tar pit fossil collection includes Juniperus (C3) wood specimens that 14C date between 7.7 and 55 thousand years (kyr) B.P., providing a constrained record of plant response for southern California during the last glacial period. Atmospheric CO2 concentration ([CO2]) ranged between 180 and 220 ppm during glacial periods, rose to approximately 280 ppm before the industrial period, and is currently approaching 380 ppm in the modern atmosphere. Here we report on delta13C of Juniperus wood cellulose, and show that glacial and modern trees were operating at similar leaf-intercellular [CO2](ci)/atmospheric [CO2](ca) values. As a result, glacial trees were operating at ci values much closer to the CO2-compensation point for C3 photosynthesis than modern trees, indicating that glacial trees were undergoing carbon starvation. In addition, we modeled relative humidity by using delta18O of cellulose from the same Juniperus specimens and found that glacial humidity was approximately 10% higher than that in modern times, indicating that differences in vapor-pressure deficits did not impose additional constrictions on ci/ca in the past. By scaling ancient ci values to plant growth by using modern relationships, we found evidence that C3 primary productivity was greatly diminished in southern California during the last glacial period.

Atmosphere↗
PubMed1994

TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR.

A cDNA encoding a double-stranded-RNA (dsRNA)-binding protein was isolated by screening a HeLa cell cDNA expression library for proteins that bind the HIV-1 Rev-responsive-element RNA. The cDNA encoded a protein that was identical to TRBP, the previously reported cellular protein that binds the transactivation response element (TAR) RNA of human immunodeficiency virus type 1. TRBP inhibited phosphorylation of the interferon-induced ribosome-associated protein kinase PKR and of the eukaryotic translation initiation factor eIF-2 alpha in a transient-expression system in which the translation of a reporter gene was inhibited by the localized activation of PKR. TRBP expression in HeLa cells complemented the growth and protein-synthesis defect of a vaccinia virus mutant lacking the expression of the dsRNA-binding protein E3L. These results implicate TRBP as a cellular regulatory protein that binds RNAs containing specific secondary structure(s) to mediate the inhibition of PKR activation and stimulate translation in a localized manner.

Animals↗
PubMed1995

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

Amino Acid Sequence↗
PubMed2001

HIV-1 Tat protein interacts with mammalian capping enzyme and stimulates capping of TAR RNA.

HIV gene expression is subject to a transcriptional checkpoint, whereby negative transcription elongation factors induce an elongation block that is overcome by HIV Tat protein in conjunction with P-TEFb. P-TEFb is a cyclin-dependent kinase that catalyzes Tat-dependent phosphorylation of Ser-5 of the Pol II C-terminal domain (CTD). Ser-5 phosphorylation confers on the CTD the ability to recruit the mammalian mRNA capping enzyme (Mce1) and stimulate its guanylyltransferase activity. Here we show that Tat spearheads a second and novel pathway of capping enzyme recruitment and activation via a direct physical interaction between the C-terminal domain of Tat and Mce1. Tat stimulates the guanylyltransferase and triphosphatase activities of Mce1 and thereby enhances the otherwise low efficiency of cap formation on a TAR stem-loop RNA. Our findings suggest that multiple mechanisms exist for coupling transcription elongation and mRNA processing.

Animals↗
PubMed2002

Tat and trans-activation-responsive (TAR) RNA-independent induction of HIV-1 long terminal repeat by human and murine cyclin T1 requires Sp1.

P-TEFb, cyclin T1 + CDK9, is needed for the expression of cellular promoters and primate lentiviral long terminal repeats (LTRs). Curiously, cellular and lentiviral promoters differ dramatically in the requirements for positive transcriptional elongation factor (P-TEF) b activity. Lentiviral LTRs, but not cellular promoters, need an RNA-associated P-TEFb/Tat/TAR (trans-activation-responsive) RNA ternary complex. Ternary complex defective murine cycT1 is apparently inactive for lentiviral transcription. Why P-TEFb requires Tat/TAR for LTRs but not for cellular promoters remains unknown. To explore this question, we sought to determine whether DNA targeting of murine and human cyclin T1 can reconstitute a Tat/TAR-independent activity to the HIV-1 LTR. In the absence of Tat and TAR, we found that both HuCycT1 and MuCycT1 can robustly activate the HIV-1 LTR. We further showed that Sp1 is necessary and sufficient for this DNA-targeted activity. Thus, like cellular promoters, HIV-1 LTR can use P-TEFb function without a Tat/TAR RNA complex. This activity could explain recent findings of robust HIV-1 replication in rat cells that cannot form a P-TEFb/Tat/TAR moiety.

Animals↗
PubMed1992

A molecular mechanics investigation of RNA complexes. I. Ethidium intercalation in an HIV-1 TAR RNA sequence with an unpaired adenosine.

Nucleic acid complexes with ethidium intercalated into different sites in a segment of HIV-1 TAR RNA with an unpaired A base, along with corresponding complexes with a normal RNA sequence without an unpaired base were studied by molecular mechanics energy minimization methods. Different intercalation geometries as well as different orientations of the ethidium molecule in the intercalation sites were tested. A general binding affinity enhancement for the ethidium binding to the bulge sequence compared with the normal RNA segment was obtained. With the unpaired adenosine base stacked in the duplex, the binding site adjacent to the 3' side of the bulge was found to be the most energetically favorable binding site, and the intercalation site 5' to the bulge in the same sequence is much less favorable. Unique correlated backbone conformational changes on binding of ethidium to the intercalation site 3' to the bulge were found to relieve backbone strains caused by the stacking of the unpaired base into the helix. These backbone conformational changes present a plausible molecular basis for the experimentally observed ethidium binding preference in this bulge RNA segment (L.S. Ratmeyer, R. Vinayak, G. Zon and W.D. Wilson, J. Med. Chem. 35, 966, 1992).

Base Sequence↗