Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “TARS”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 397 records · Page 22Linked to original sources
PubMed2005

Arginine mutations within a transmembrane domain of Tar, an Escherichia coli aspartate receptor, can drive homodimer dissociation and heterodimer association in vivo.

The interactions between the TM (transmembrane) domains of many membrane proteins are important for their proper functioning. Mutations of residues into positively charged ones within TM domains were reported to be involved in many genetic diseases, possibly because these mutations affect the self- and/or hetero-assembly of the corresponding proteins. To our knowledge, despite significant progress in understanding the role of various amino acids in TM-TM interactions in vivo, the direct effect of positively charged residues on these interactions has not been studied. To address this issue, we employed the N-terminal TM domain of the aspartate receptor (Tar-1) as a dimerization model system. We expressed within the ToxR TM assembly system several Tar-1 constructs that dimerize via polar- or non-polar amino acid motifs, and mutated these by replacement with a single arginine residue. Our results have revealed that a mutation in each of the motifs significantly reduced the ability of the TMs to dimerize. Furthermore, a Tar-1 construct that contained two arginine residues was unable to correctly integrate itself into the membrane. Nevertheless, an exogenous synthetic Tar-1 peptide containing these two arginine residues was able to inhibit in vivo the marked dimerization of a mutant Tar-1 construct that contained two glutamate residues at similar positions. This indicates that hetero-assembly of TM domains can be mediated by the interaction of two oppositely charged residues, probably by formation of ion pairs. This study broadens our knowledge regarding the effect of positively charged residues on TM-TM interactions in vivo, and provides a potential therapeutic approach to inhibit uncontrolled dimerization of TM domains caused by mutations of polar amino acids.

Amino Acid Motifs↗
PubMed2002

Loop-loop interaction of HIV-1 TAR RNA with N3'-->P5' deoxyphosphoramidate aptamers inhibits in vitro Tat-mediated transcription.

A hairpin RNA aptamer has been identified by in vitro selection against the transactivation-responsive element (TAR) of HIV-1. A nuclease-resistant N3' --> P5' phosphoramidate isosequential analog of this aptamer also folds as a hairpin and forms with TAR a loop-loop "kissing" complex with a binding constant in the low nanomolar range as demonstrated by electrophoretic mobility-shift assays and surface plasmon resonance experiments. The key structural determinants, which contribute to the stability of the RNA aptamer-TAR complex, loop complementarity and the GA residues closing the aptamer loop, remain crucial for the N3' --> P5' aptamer-TAR complex. Moreover, the N3' --> P5' phosphoramidate aptamer specifically interferes with the binding of a peptide derived from the transactivator protein (Tat) peptide to TAR and selectively inhibits the Tat-mediated transcription in an in vitro assay, which marks this nuclease-resistant aptamer as a relevant candidate for experiments in cells.

Amides↗
PubMed1991

The number of positively charged amino acids in the basic domain of Tat is critical for trans-activation and complex formation with TAR RNA.

The basic domain of Tat is required for trans-activation of viral gene expression. We have performed scanning peptide studies to demonstrate that only this domain is capable of binding to the TAR RNA stem-loop. Strikingly, the basic domain of the other human immunodeficiency virus trans-acting factor, Rev, but no other region, is also capable of binding to TAR. Peptide derivatives of Tat do not require the highly conserved glutamine residue at position 54 for TAR binding, since it may be substituted or deleted. In addition, the two lysine residues may be replaced by arginines. Analysis of binding and trans-activation demonstrated that homopolymers of arginine can completely substitute for the basic domain. Such homopolymers have high affinity for wild-type TAR RNA and lower affinity for mutant TAR. Homopolymers of six to nine arginines substituting for the basic domain of Tat enable full trans-activation in vivo. Homopolymers of at least seven arginines are required for detectable in vitro complex formation, although approximately 30% trans-activation is achieved with a mutant Tat containing only five arginines.

Amino Acid Sequence↗
PubMed2002

CheW binding interactions with CheA and Tar. Importance for chemotaxis signaling in Escherichia coli.

The initial signaling events underlying the chemotactic response of Escherichia coli to aspartic acid occur within a ternary complex that includes Tar (an aspartate receptor), CheA (a protein kinase), and CheW. Because CheW can bind to CheA and to Tar, it is thought to serve as an adapter protein in this complex. The functional importance of CheW binding interactions, however, has not been investigated. To better define the role of CheW and its binding interactions, we performed biochemical characterization of six mutant variants of CheW. We examined the ability of the purified mutant CheW proteins to bind to CheA and Tar, to promote formation of active ternary complexes, and to support chemotaxis in vivo. Our results indicate that mutations which eliminate CheW binding to Tar (V36M) or to CheA (G57D) result in a complete inability to form active ternary complexes in vitro and render the CheW protein incapable of mediating chemotaxis in vivo. The in vivo signaling pathway can, however, tolerate moderate changes in CheW-Tar and CheW-CheA affinities observed with several of the mutants (G133E, G41D, and 154ocr). One mutant (R62H) provided surprising results that may indicate a role for CheW in addition to binding CheA/receptors and promoting ternary complex formation.

Anisotropy↗
PubMed2003

The apical loop of the HIV-1 TAR RNA hairpin is stabilized by a cross-loop base pair.

The TAR hairpin of the HIV-1 RNA genome is indispensable for trans-activation of the viral promoter and virus replication. The TAR structure has been studied extensively, but most attention has been directed at the three-nucleotide bulge that constitutes the binding site of the viral Tat protein. In contrast, the conformational properties of the apical loop have remained elusive. We performed biochemical studies and molecular dynamics simulations, which indicate that the TAR loop is structured and stabilized by a cross-loop base pair between residues C30 and G34. Mutational disruption of the cross-loop base pair results in reduced Tat response of the LTR promoter, which can be rescued by compensatory mutations that restore the base pair. Thus, Tat-mediated transcriptional activation depends on the structure of the TAR apical loop. The C30-G34 cross-loop base pair classes TAR in a growing family of hairpins with a structured loop that was recently identified in ribosomal RNA, tRNA, and several viral and cellular mRNAs.

2-Aminopurine↗
PubMed2004

Poly(ADP-ribose) polymerase-1 is a negative regulator of HIV-1 transcription through competitive binding to TAR RNA with Tat.positive transcription elongation factor b (p-TEFb) complex.

Human immunodeficiency virus, type 1 (HIV-1) transcription is regulated by a virus-encoded protein, Tat, which forms a complex with a host cellular factor, positive transcription elongation factor b (P-TEFb). When this complex binds to TAR RNA synthesized from the HIV-1 long terminal repeat promoter element, transcription is trans-activated. In this study we showed that, in host cells, HIV-1 transcription is negatively regulated by competition of poly(ADP-ribose) polymerase-1 (PARP-1) with Tat.P-TEFb for binding to TAR RNA. PARP-1, which has a high affinity for TAR RNA (K(D) = 1.35 x 10(-10) M), binds to the loop region of TAR RNA and displaces Tat or Tat.P-TEFb from the RNA. In vitro transcription assays showed that this displacement leads to suppression of Tat-mediated trans-activation of transcription. Furthermore in vivo expression of luciferase or destabilized enhanced green fluorescent protein genes under the control of the HIV-1 long terminal repeat promoter was suppressed by PARP-1. Thus, these results suggest that PARP-1 acts as a negative regulator of HIV-1 transcription through competitive binding with Tat or the Tat.P-TEFb complex to TAR RNA.

Binding, Competitive↗
PubMed2002

In vitro selection of DNA aptamers against the HIV-1 TAR RNA hairpin.

In vitro selection was performed to identify DNA aptamers against the TAR RNA stem-loop structure of HIV-1. A counterselection step allowed the elimination of kissing complex-forming aptamers previously selected (Boiziau et al. J. Biol. Chem. 1999; 274:12730). This led to the emergence of oligonucleotides, most of which contained two consensus sequences, one targeted to the stem 3'-strand (5'-CCCTAGTTA) and the other complementary to the TAR apical loop (5'-CTCCC). The best aptamer could be shortened to a 19-mer oligonucleotide, characterized by a dissociation constant of 50 nM. A 16-mer oligonucleotide complementary to the TAR stem 3'-strand could also be derived from the identified aptamers, with an equal affinity (Kd = 50 nM). Experiments performed to elucidate the interaction between TAR and the aptamers (UV melting measures, enzymatic and chemical footprints) demonstrated that the TAR stem 5'-strand was not simply displaced as a result of the complex formation but unexpectedly remained associated on contact with the antisense oligonucleotide. We suggest that a multistranded structure could be formed.

DNA, Antisense↗
PubMed1995

Molecular cloning and characterization of a TAR-binding nuclear factor from T cells.

The TAt protein of the human immunodeficiency virus type 1 (HIV-1) activates the expression of viral mRNA through a cis-acting element in the LTR termed TAR. TAR RNA forms a stable stem-loop structure. Mutagenesis studies indicate that the stem structure, the primary sequence of the loop, and three unpaired bases in the stem (bulge) are important for Tat activation. Using the in vitro-transcribed TAR RNA as a probe, we have cloned a gene (TARBP-b) that encodes a TAR-binding protein from a cDNA expression library derived from Hut-78 cells. Expression of the 1.4-kb TARBP-b mRNA was observed in all mammalian cell lines tested. TARBP-b binds specifically to the bulge region of TAR RNA and trans-activates the HIV-1 long terminal repeat in the presence of ptat and prev expression plasmids. These results suggest that TARBP-b contributes to tat-mediated trans-activation.

Amino Acid Sequence↗
PubMed1992

Structural features in TAR RNA of human and simian immunodeficiency viruses: a phylogenetic analysis.

A comparative analysis of TAR RNA structures in human and simian immunodeficiency viruses reveals the conservation of certain structural features despite the divergence in sequence. Both the TAR elements of HIV-1 and SIV-chimpanzee can be folded into relatively simple one-stem hairpin structures. Chemical and RNAase probes were used to analyze the more complex structure of HIV-2 TAR RNA, which folds into a branched hairpin structure. A surprisingly similar RNA conformation can be proposed for SIV-mandrill, despite considerable divergence in nucleotide sequence. A third structural presentation of TAR sequences is seen for SIV-african green monkey. These results are generally consistent with the classification of HIV-SIV viruses in four subgroups based on sequence analyses (both nucleotide- and amino acid-sequences). However, some conserved TAR structures were detected for members of different virus subgroups. It is therefore proposed that RNA structure analysis might provide an additional tool for determining phylogenetic relationships among the HIV-SIV viruses.

Base Composition↗
PubMed1993

Design and synthesis of RNA miniduplexes via a synthetic linker approach. 2. Generation of covalently closed, double-stranded cyclic HIV-1 TAR RNA analogs with high Tat-binding affinity.

We recently developed an approach which allows rapid generation of short, double-stranded oligonucleotides whereby one end of the duplex was joined and stabilized by a synthetic linker of specific design (miniduplexes)(6). Model miniduplexes based on the HIV-1 TAR RNA hairpin were shown to be thermodynamically stable and good substrates for binding by the HIV-1 Tat protein which normally bind to natural TAR (6). In this study, we have extended our studies to the design, synthesis and analysis of the binding properties of covalently closed, double-stranded, cyclic RNA miniduplexes. A strategy using automated chemical synthesis and T4 RNA ligase-catalyzed cyclization was employed to generate cyclic oligoribonucleotides. When both ends of a shortened, wild-type TAR RNA stem (9 bp) were covalently linked through either nucleotidic loops (4-6 nt) or synthetic linkers (derivatized from hexaethylene glycol), the resulting cyclic TAR RNA analogs were good substrates for binding by both Tat-derived peptide or full-length Tat protein. Interestingly, the cyclic TAR analogs failed to show any binding if the synthetic linker was reduced in length (e.g. derivatized from triethylene glycol), although such linkers are acceptable in the hairpin-shaped miniduplexes series (6). This implies that RNA conformational changes are required for Tat binding and that these changes are restricted in certain cyclic variants. Our findings suggest that covalently-closed nucleic acid miniduplexes may be useful both to study nucleic acid-protein interactions as well as to provide a basis for therapeutic intervention as transcription decoys.

Amino Acid Sequence↗
PubMed2002

The bulge region of HIV-1 TAR RNA binds metal ions in solution.

Binding of Mg2+, Ca2+ and Co(NH3)6(3+) ions to the HIV-1 TAR RNA in solution was analysed by 19F NMR spectroscopy, metal ion-induced RNA cleavages and Brownian dynamics (BD) simulations. Chemically synthesised 29mer oligoribonucleotides of the TAR sequence labelled with 5-fluorouridine (FU) were used for 19F NMR-monitored metal ion titration. The chemical shift changes of fluorine resonances FU-23, FU-25 and FU-40 upon titration with Mg2+ and Ca2+ ions indicated specific, although weak, binding at the bulge region with the dissociation constants (K(d)) of 0.9 +/- 0.6 and 2.7 +/- 1.7 mM, respectively. Argininamide, inducing largest (19)F chemical shifts changes at FU-23, was used as a reference ligand (K(d) = 0.3 +/- 0.1 mM). In the Pb2+-induced TAR RNA cleavage experiment, strong and selective cleavage of the C24-U25 phosphodiester bond was observed, while Mg2+ and Ca2+ induced cuts at all 3-nt residues of the bulge. The inhibition of Pb2+-specific TAR cleavage by di- and trivalent metal ions revealed a binding specificity [in the order Co(NH3)6(3+) > Mg2+ > Ca2+] at the bulge site. A BD simulation search of potential magnesium ion sites within the NMR structure of HIV-1 TAR RNA was conducted on a set of 20 conformers (PDB code 1ANR). For most cases, the bulge region was targeted by magnesium cations.

Arginine↗
PubMed2004

LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1.

One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera-TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets.

Base Sequence↗
PubMed1993

HIV-1 TAR RNA-binding proteins control TAT activation of translation in Xenopus oocytes.

Human immunodeficiency virus (HIV-1) gene expression is activated by the viral TAT protein that interacts with an RNA sequence, TAR, located at the 5' end of all viral mRNAs. TAT functions primarily as a transcriptional activator in mammalian cells. However, in Xenopus oocytes TAT functions primarily as a translational activator. TAR is an RNA structure comprising a partially base-paired stem, a tripyrimidine bulge in the upper stem, and an unpaired six-nucleotide loop. In vitro, TAT binds directly to the bulge with no requirement for the loop. In vivo, however, mutations in the loop abolish TAT activation of transcription and translation, implying a requirement for TAR-binding cellular factors. We now provide genetic evidence for the presence of two TAR-specific cellular factors in Xenopus oocytes. These factors display independent and mutually exclusive interactions with either the loop or the bulge region of TAR. Furthermore, by using in vivo RNA competition assays we show that the cellular factors regulate the accessibility of the TAT binding site. The fact that Xenopus oocytes contain factors that specifically interact with a human viral RNA sequence might indicate that the TAT/TAR interaction is subverting a conserved pathway in the cell.

Animals↗
PubMed1994

Crystallization studies of the human immunodeficiency virus (HIV-1) Tat protein and its trans-activation response element (TAR) RNA.

Small single crystals are reported of a complex between a small peptide fragment of the HIV-1 Tat protein and a fragment of the RNA to which it binds. Tat is responsible for enhancing the level of expression of the human immunodeficiency virus type 1 (HIV-1) and is a logical target for AIDS therapy. Tat may function to increase the level of transcription initiation or to prevent premature termination of transcripts. In vitro, Tat binds through its basic domain (two Lys and six Arg in nine residues) to a three-nucleotide bulge of a stem-loop RNA structure called TAR. Complex formation between Tat and TAR is necessary for Tat activity. Peptides which contain the basic region of Tat also bind to TAR RNA. We have carried out crystallization experiments on a 27-nucleotide fragment of TAR RNA and on complexes between two Tat peptides and TAR.

Journal Article↗
PubMed1986

Mutations in tar suppress defects in maltose chemotaxis caused by specific malE mutations.

Maltose-binding protein (MBP), which is encoded by the malE gene, is the maltose chemoreceptor of Escherichia coli, as well as an essential component of the maltose uptake system. Maltose-loaded MBP is thought to initiate a chemotactic response by binding to the tar gene product, the signal transducer Tar, which is also the aspartate chemoreceptor. To study the interaction of MBP with Tar, we selected 14 malE mutants which had specific defects in maltose taxis. Three of these mutants were fully active in maltose transport and produced MBP in normal amounts. The isoelectric points of the MBPs from these three mutants were identical to (malE461 and malE469) or only 0.1 pH unit more basic than (malE454) the isoelectric point of the wild-type protein (pH 5.0). Six of the mutations, including malE454, malE461, and malE469, were mapped in detail; they were located in two regions within malE. We also isolated second-site suppressor mutations in the tar gene that restored maltose taxis in combination with the closely linked malE454 and malE461 mutations but not with the malE469 mutation, which maps in a different part of the gene. This allele-specific suppression confirmed that MBP and Tar interact directly.

ATP-Binding Cassette Transporters↗
PubMed1988

Purification and characterization of the wild-type and mutant carboxy-terminal domains of the Escherichia coli Tar chemoreceptor.

The carboxy-terminal half of the Escherichia coli Tar chemoreceptor protein was cloned into an overproducing plasmid with the transcription of the insert under the control of the strong hybrid tac promoter. Two dominant mutations in the tar gene, which result in "tumble-only" (tar-526) or "swim-only" (tar-529) phenotypes and which are postulated to produce proteins locked in specific signalling modes, were introduced separately onto the overproducing plasmid. After induction with isopropyl-beta-D-thiogalactopyranoside, cells containing the plasmids produced about 10% of their soluble cellular protein as the carboxy-terminal fragments. A scheme to purify the overproduced fragments was developed. Typical yields of pure fragment were 5, 30, and 20 mg per liter of induced culture for the wild type, 526 mutant, and 529 mutant, respectively. Fast-protein liquid chromatography-gel filtration analysis of the pure fragments showed that they all existed as oligomers (ca. 103,000 daltons), possibly trimers or tetramers (monomer size is 31,000 daltons). However, the 529 mutant fragment showed an additional oligomeric form (240,000 daltons) corresponding approximately to an octamer. When chromatographed in the presence of 1% octylglucoside, all three fragments showed an identical single oligomeric size of about 135,000 daltons. Further differences between the fragments such as ion-exchange behavior and susceptibility to degradation were found. Taken together, these results suggest that conformational differences between the 529 mutant fragment and the other fragments exist and that these differences may correlate with the phenotypic effects of the tar-529 mutation.

Bacterial Proteins↗
PubMed1992

Human immunodeficiency virus type 1 Tat-mediated trans activation correlates with the phosphorylation state of a cellular TAR RNA stem-binding factor.

Protein kinase C (PKC) is involved in the mitogenic stimulation of cell proliferation and has recently been reported to be essential for Tat-mediated trans activation. We have determined that RNA binding of a cellular factor which specifically interacts with the trans-activation response region (TAR) is blocked in cells depleted of PKC activity by chronic phorbol myristate acetate stimulation. We also show that nuclear extracts can be depleted of the cellular TAR-binding factor by in vitro treatment with purified protein phosphatase 2A. Furthermore, TAR RNA-binding activity can be partially restored to depleted nuclear extracts in vitro by addition of PKC. Chimeric constructs in which the Tat protein is artificially tethered to viral RNA show PKC independence for Tat-mediated trans activation. Specific mutations in the TAR RNA stem region which cause reduced binding of host cell factor in vitro also cause reduced Tat-mediated trans activation in vivo. Together, these results suggest that phosphorylation-dependent binding of a cellular cofactor to TAR RNA is an essential step in Tat-mediated trans activation. Deciphering the regulation of Tat-mediated trans activation by phosphorylation will be critical in fully understanding the regulation of human immunodeficiency virus type 1 activation.

Base Sequence↗
PubMed1993

TAR RNA binding properties and relative transactivation activities of human immunodeficiency virus type 1 and 2 Tat proteins.

Using gel shift assays, we found that the human immunodeficiency virus type 1 (HIV-1) Tat protein (Tat-1) bound both HIV-1 and HIV-2 TAR RNAs with similar high affinities. In contrast, the HIV-2 Tat protein (Tat-2) bound only TAR-2 RNA with high affinity. We conclude that the weak in vivo activity of Tat-2 on the HIV-1 long terminal repeat that has been observed previously is likely the result of low affinity for TAR-1 RNA. Additionally, TAR-2 RNA was found to contain multiple specific binding sites for Tat proteins. GAL4-Tat fusion proteins were analyzed to compare the relative transactivation activities of Tat-1 and Tat-2 in the absence of requirements for binding to TAR RNAs. The GAL4-Tat-2 protein was found to transactivate synthetic promoters containing GAL4 binding sites at levels severalfold higher than did the GAL4-Tat-1 protein.

Amino Acid Sequence↗