CXCR3 internalization following T cell-endothelial cell contact: preferential role of IFN-inducible T cell alpha chemoattractant (CXCL11).

Chemokine receptors are rapidly desensitized and internalized following ligand binding, a process that attenuates receptor-mediated responses. However, the physiological settings in which this process occurs are not clear. Therefore, we examined the fate of CXCR3, a chemokine receptor preferentially expressed on activated T cells following contact with endothelial cells. By immunofluorescence microscopy and flow cytometry, we found that CXCR3 was rapidly internalized when T cells were incubated with IFN-gamma-activated human saphenous vein endothelial cells (HSVEC), but not with resting HSVEC. Similar results were obtained using human CXCR3-transfected murine 300-19 B cells. CXCR3 down-regulation was significantly more pronounced when T cells were in contact with HSVEC than with their supernatants, suggesting that CXCR3 ligands were efficiently displayed on the surface of HSVEC. Using neutralizing mAbs to IFN-induced protein-10 (CXCL10), monokine induced by IFN-gamma (CXCL9), and IFN-inducible T cell alpha chemoattractant (I-TAC; CXCL11), we found that even though I-TAC was secreted from IFN-gamma-activated HSVEC to lower levels than IFN-induced protein-10 or the monokine induced by IFN-gamma, it was the principal chemokine responsible for CXCR3 internalization. This correlated with studies using recombinant chemokines, which revealed that I-TAC was the most potent inducer of CXCR3 down-regulation and of transendothelial migration. Known inhibitors of chemokine-induced chemotaxis, such as pertussis toxin or wortmannin, did not reduce ligand-induced internalization, suggesting that a distinct signal transduction pathway mediates internalization. Our data demonstrate that I-TAC is the physiological inducer of CXCR3 internalization and suggest that chemokine receptor internalization occurs in physiological settings, such as leukocyte contact with an activated endothelium.

Expression of the Na(+)/Ca(2+) exchanger ameliorates ionomycin-induced cell death.

PC12 cells were stably transfected with cDNA encoding the Na(+)/Ca(2+) exchanger (NCX1.4). A robust Na(+)-dependent Ca(2+) uptake confirmed the functional expression of the protein. When NCX1. 4 expressing cells (NO) and vector transfected control cells (VC) were exposed to 0.5-20 microM ionomycin for 6 h, a dose-dependent increase in LDH release was observed. LDH release was significantly reduced in NO when compared with VC. When either VC and NO were treated with 3 microM ionomycin and 1.1 mM EGTA, the increase in LDH release was nearly abolished. However, when VC and NO were treated with ionomycin and then EGTA was added 2 min later, LDH release remained elevated. These data suggest ionomycin-induced cell death was Ca(2+) dependent and expressing NCX1.4 may have ameliorated cell death by reducing elevated [Ca(2+)](I).

Zinc transport in the brain: routes of zinc influx and efflux in neurons.

Studies of the routes of entry and exit for zinc in different tissues and cell types have shown that zinc can use several pathways of exit or entry. In neurons, known pathways include (1) presynaptic release along with glutamate when synaptic vesicles empty their contents into the synaptic cleft, (2) voltage-gated L-type Ca(2+) channels and glutamate-gated channels that provide an entry route when cells are depolarized and that mediate extracellular zinc toxicity and (3) a plasma membrane transporter potentially present in all neurons important for cellular zinc homeostasis. The least understood of these pathways, in terms of mechanism, is the transporter pathway. The kinetics of zinc uptake in cultured neurons under resting conditions are consistent with and suggest the existence of a saturable transporter in the plasma membrane. The proteins responsible for plasma membrane zinc transport have not yet been definitely identified. Likely candidates include two proteins identified by molecular cloning termed zinc transporter 1 and divalent cation transporter DCT1. Both proteins have been shown to be expressed in the brain, but only DCT1 is clearly demonstrated to be a transport protein, whereas zinc transporter 1 may only modulate zinc transport in association with as-yet-unidentified proteins. Understanding the mechanism and neuromodulation of plasma membrane zinc transport will be an important first step toward a complete understanding of neuronal zinc homeostasis.

Evidence for a zinc/proton antiporter in rat brain.

The data presented in this paper are consistent with the existence of a plasma membrane zinc/proton antiport activity in rat brain. Experiments were performed using purified plasma membrane vesicles isolated from whole rat brain. Incubating vesicles in the presence of various concentrations of 65Zn2+ resulted in a rapid accumulation of 65Zn2+. Hill plot analysis demonstrated a lack of cooperativity in zinc activation of 65Zn2+ uptake. Zinc uptake was inhibited in the presence of 1 mM Ni2+, Cd2+, or CO2+. Calcium (1 mM) was less effective at inhibiting 65Zn2+ uptake and Mg2+ and Mn2+ had no effect. The initial rate of vesicular 65Zn2+ uptake was inhibited by increasing extravesicular H+ concentration. Vesicles preloaded with 65Zn2+ could be induced to release 65Zn2+ by increasing extravesicular H+ or addition of 1 mM nonradioactive Zn2+. Hill plot analysis showed a lack of cooperativity in H+ activation of 65Zn2+ release. Based on the Hill analyses, the stoichiometry of transport may include Zn2+/Zn2+ exchange and Zn2+/H+ antiport, the latter being potentially electrogenic. Zinc/proton antiport may be an important mode of zinc uptake into neurons and contribute to the reuptake of zinc to replenish presynaptic vesicle stores after stimulation.

Zinc inhibits Ca2+ transport by rat brain NA+/Ca2+ exchanger.

Calcium transport by the Na+/Ca2+ exchanger was measured in plasma membranes vesicles purified from rat brain and in primary rat cortical cell culture. Sodium-loaded vesicles rapidly accumulate Ca2+ via Na+/Ca2+ exchange (Na+(i)-dependent Ca2+ uptake). Extravesicular zinc inhibited Na+/Ca2+ exchange as evidenced by a reduction of the initial velocity of Ca2+ uptake. Significant inhibition of Ca2+ uptake was seen at concentrations of zinc as low as 3 microM. Lineweaver-Burk analysis of the data was consistent with noncompetitive inhibition with respect to extravesicular Ca2+ concentration. The Ki for zinc inhibition of Ca2+ uptake determined from a Dixon plot was 14.5 microM. This is within the range of zinc concentrations thought to be obtained extracellularly after excitation. When vesicles were preloaded with Ca2+, extravesicular zinc also inhibited reversal of Na+/Ca2+ exchange (Na+(i)-dependent Ca2+ release) although its potency was much less: concentrations of > or = 30 microM zinc were required. Zinc inhibition of Ca2+ release was not Na+ dependent. Na+(i)-dependent calcium uptake by rat cortical cells in primary culture also was inhibited by zinc. The extent of inhibition was similar to that seen for inhibition of Na+(i)-dependent Ca2+ uptake in membrane vesicles, but the potency was less. The results suggest that Ca2+ transport by the Na+/Ca2+ exchanger is inhibited by concentrations of zinc thought to be attained extracellularly after excitation.

Characterization of a plasma membrane zinc transporter in rat brain.

Many studies now show that zinc plays a critical and unique role in central nervous system development and function. The cellular mechanisms of zinc efflux and influx are largely unknown and few models exist that describe cellular zinc transport in the brain. This report provides convincing evidence of a zinc transporter in plasma membrane vesicles isolated from rat brain. Zinc uptake was saturable (Km = 15 microM; Vmax = 10 nmol/mg per 30 s), was seen in the absence of ATP, and was unaffected by gradients for other ions such as Na+ or K+. Increasing the ionic strength of the extravesicular media with Na+, K+, or choline+ reduced zinc uptake approximately 50%. Whereas, increasing extravesicular H+ concentration (pH = 5) resulted in near complete inhibition of zinc uptake. Intravesicular zinc was rapidly released upon lowering extravesicular concentrations of zinc with the heavy metal chelator O-phenanthroline (1 mM). The results are consistent with a freely-reversible transport of zinc across the plasma membrane of neurons.

Regional differences in expression of transcripts for Na+/Ca2+ exchanger isoforms in rat brain.

The Na+/Ca2+ exchanger has a primary role in maintaining intraneuronal Ca2+ homeostasis. There are three distinct Na+/Ca2+ exchanger isoforms cloned from rat brain, NCX1, NCX2 and NCX3, which are the products of three different genes. In the present study, isoform expression in different regions of rat brain was determined by using reverse transcription PCR (RT-PCR) and Northern analysis. RT-PCR detected all three Na+/Ca2+ exchanger isoforms in each region studied (brainstem/spinal cord, cerebellum, cerebral cortex, striatum/septum and hippocampus). Northern analysis was performed to determine the steady-state mRNA levels of each isoform. NCX1 had two transcripts, 14 and 7 kb, and the 7-kb transcript was predominant in brainstem/spinal cord, cerebellum and hippocampus. NCX2 expression (4.8-kb transcript) was an order of magnitude higher than NCX1 or NCX3 expression in all the five areas except brainstem/spinal cord where the 4.8-kb transcript was nearly absent. The third isoform (NCX3) had two transcripts, one was 6 kb and the other was 4 kb. The 6-kb transcript was predominant in brainstem/spinal cord and cerebellum. The results suggest that Na+/Ca2+ exchanger isoforms are expressed ubiquitously in rat brain but that each isoform shows a unique distribution within the brain. The exchanger probably participates in the regulation of intracellular calcium homeostasis in a wide range of cell types within the brain. Furthermore, individual cells may contain more than one type of exchanger isoform with distinct subcellular distributions.

Alzheimer's amyloid-beta peptide inhibits sodium/calcium exchange measured in rat and human brain plasma membrane vesicles.

Na+/Ca2+ exchange activity was measured by monitoring vesicular Ca2+ content after incubation in buffers containing 45Ca2+. When Na+-loaded vesicles were placed into Na+-free buffer, vesicular Ca2+ content increased rapidly and reached a plateau after two to three minutes. Only preaggregated amyloid-beta1-40 (Abeta1-40) and Abeta25-35 reduced vesicular Ca2+ content. Both peptides produced a maximal reduction in Ca2+ content of approximately 50%. The peptides reduced Ca2+ content with similar potency and half maximal effects were seen at less than 10 microM for Abeta25-35. Calcium-loaded vesicles mediate a rapid Ca2+/Ca2+ exchange, which also was inhibited by aggregated Abeta25-35. Aggregated Abeta25-35 did not affect the passive Ca2+ permeability of the vesicles. Aggregated Abeta25-35 reduced Ca2+ content in plasma membrane vesicles isolated from normal and Alzheimer's disease frontal cortex with less potency but the same efficacy as seen in rat brain. Aggregated Abeta25-35 did not produce nonspecific effects on vesicle morphology such as clumping or loss of intact vesicles. When placed in the buffer used to measure Ca2+ content, Congo Red at molar ratios of less than one blocked the inhibitory effect of preaggregated Abeta25-35. When added in equimolar concentrations to freshly dissolved and unaggregated Abeta25-35, Congo Red also was effective at blocking the inhibitory effect on Ca2+ content. In contrast, vitamin E (antioxidant) and N-tert-butyl-alpha-phenylnitrone (spin trapping agent) failed to block the inhibitory action of aggregated Abeta25-35. The exact mechanisms of Abeta-induced neurotoxicity in cell culture has yet to be solved. Accumulation of free radicals play a necessary role, but disruptions of Ca2+ homeostasis are also important. The data presented here are consistent with a proposed mechanism where aggregated Abeta peptides directly interact with hydrophobic surfaces of the exchanger protein and/or lipid bilayer and interfere with plasma membrane Ca2+ transport.

Genistein inhibits Na+/Ca2+ exchange activity in primary rat cortical neuron culture.

We have examined the possible regulatory effect of tyrosine kinase activity on Ca2+ transport observed in the cultured rat cortical neurons. Na+/Ca2+ exchange was studied using cells cultured for various time periods. A nearly two fold increase in Ca2+ uptake was seen when comparing 3 day and 9 day cultures. Western blot analysis also showed a two fold increase in Na+/Ca2+ exchanger (NCX1) protein levels as cells matured in culture. To study the effect of genistein (a specific tyrosine kinase inhibitor) cells were incubated with 100 microM genistein (in 1% DMSO) for 1 hour before the assay of Na+/Ca2+ exchange activity. There was a significant decrease of Ca2+ uptake in genistein treated neurons (control: 4.596+/-0.205 nmol/mg protein/15 min, n=12; genistein: 1.420+/-0.131 nmol/mg protein/15 min, n=12, mean+/-S.E. P<0.001). Daidzein, an inactive analog of genistein and phorbol myristate acetate (PMA), a PKC activator were without effect. The results suggest that as cells mature in culture, Na+/Ca2+ exchange capacity increases, as a result of greater protein expression. Exposure to genistein inhibited Ca2+ uptake suggesting that the exchanger may be modulated by tyrosine phosphorylation.

A small circular TAR RNA decoy specifically inhibits Tat-activated HIV-1 transcription.

Linear TAR RNA has previously been used as a decoy to inhibit HIV-1 transcription in vitro and HIV-1 replication in vivo. A 48 nucleotide circular RNA containing the stem, bulge and loop of the HIV-1 TAR element was synthesized using the self-splicing activity of a group I permuted intron-exon and was tested for its ability to function as a TAR decoy in vitro. This small circular TAR molecule was exceptionally stable in HeLa nuclear extracts, whereas a similar linear TAR molecule was rapidly degraded. The TAR circle bound specifically to Tfr38, a peptide containing the TAR-binding region of Tat. The ability of Tat to trans-activate transcription from the HIV-1 promoter in vitro was efficiently inhibited by circular TAR RNA but not by TAR circles that contained either bulge or loop mutations. TAR circles did not inhibit transactivation exclusively by binding to Tat since this inhibition was not reversed by adding excess Tat to the transcription reaction. Together, these data suggest that TAR circles act as decoys that inhibit transactivation by binding to Tat and at least one cellular factor. These data also demonstrate the utility of small circular RNA molecules as tools for biochemical studies.

Caloric restriction and spatial learning in old mice.

Spatial learning in old mice (19 or 24 months old), some of which had been calorically restricted beginning at 14 weeks of age, was compared to that of young mice, in two separate experiments using a Morris water maze. In the first experiment, only old mice reaching criterion performance on a cued learning task were tested in a subsequent spatial task. Thus, all old mice tested for spatial learning had achieved escape latencies equivalent to those of young controls. Despite equivalent swimming speeds, only about half the old mice in each diet group achieved criterion performance in the spatial task. In the second experiment, old and young mice all received the same number of training trials in a cued task and then in a spatial task. Immediately following spatial training, they were given a 60-s probe trial, with no platform in the pool. Both groups of old mice spent significantly less time in the quadrant where the platform had been and made significantly fewer direct crosses over the previous platform location than did the young control group. As in Experiment 1, calorie restriction failed to provide protection against aging-related deficits. However, in both experiments, some individual old mice evidenced performance in spatial learning indistinguishable from that of young controls. Separate comparisons of "age-impaired" and "age-unimpaired" old mice with young controls may facilitate the identification of neurobiological mechanisms underlying age-related cognitive decline.

Aging does not affect steady-state expression of the Na+/Ca2+ exchanger in rat brain.

1. Steady-state protein and mRNA levels of the Na+/Ca2+ exchanger were studied in old (24-month) and young (3-month) brains of male Fischer 344 rats by Western and Northern analysis. 2. Northern analysis with a cDNA proble for the Na+/Ca2+ exchanger amplified from human brain RNA indicated the presence of two transcripts for the Na+/Ca2+ exchanger (6 and 16 kb). Both transcripts were present in similar abundance in the cerebrum and hippocampus. In the cerebellum the 6-kb transcript predominated. The cerebellum had the highest overall level of expression. There were no significant age-related effects seen on the level of expression of either transcript in each of the brain areas tested. 3. Western analysis of plasma membrane vesicles purified from cerebral cortex identified a single protein of 116 kDa. Consistent with the Northern analysis, no age-related effect on protein levels was seen. 4. The mechanisms underlying altered Na+/Ca2+ exchange activity in aging rat brain (Michaelis, 1989) most likely do not involve changes in gene expression and are therefore more likely to represent posttranslation modifications of the protein.

Post-transcriptional regulation of the Na+/Ca2+ exchanger in aging rat heart.

Altered calcium homeostasis in the senescent heart appears to be the result, at least in part, of decreased Na+/Ca2+ exchange activity. To further investigate the basis of the decrease in Na+/Ca2+ exchange activity, Na+/Ca2+ exchanger gene expression in the heart was compared in 3 and 24 month old male Fischer 344 rats. Sarcolemmal vesicles prepared from left ventricle and septum showed reduced Na(+)-dependent Ca2+ uptake in 24 month old animals when compared to 3 month old animals (0.156 +/- 0.005 and 0.135 +/- 0.008 nmol Ca2+/mg/10 s; mean +/- S.E. for 3 month and 24 month old animals, respectively). Western analysis showed immunodetectable Na+/Ca2+ exchanger protein levels were decreased by 19% in 24 month old animals when compared to 3 month old animals. Poly(A+) RNA was purified from left and right ventricle and left and right atria and subjected to Northern analysis using digoxin labeled cDNA probes for the Na+/Ca2+ exchanger and actin. The Na+/Ca2+ exchanger probe labeled a 7 kb message in both ventricle and atria, while the actin probe labeled both beta-actin (2.2 kb) and alpha-actin (1.4 kb). The steady state level of expression of Na+/Ca2+ exchanger Poly(A+) RNA when normalized to beta-actin, was similar when ventricle and atria were compared. There were no observable differences in Na+/Ca2+ exchanger or alpha-actin Poly(A+) RNA steady state levels when comparing 3 and 24 month old animals. The results suggest that reduced Na+/Ca2+ exchange activity in the left ventricle of 24 month old animals was most likely the result of post-transcriptional modification of the protein that was detectable by Western analysis.

Characterization of exchange inhibitory peptide effects on Na+/Ca2+ exchange in rat and human brain plasma membrane vesicles.

The inhibitory effects of Na+/Ca2+ exchange inhibitory peptide (XIP), which corresponds to residues 219-238 of the Na+/Ca2+ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na(+)-dependent Ca2+ uptake in both rat brain [IC50 = 3.05 +/- 0.69 microM (mean +/- SE)] and human brain (IC50 = 3.58 +/- 0.58 microM). The maximal inhibition seen in rat brain vesicles was approximately 80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na(+)-dependent Ca2+ release, and the inhibitory effect was enhanced by increasing the extravesicular Na+ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na+. When XIP was added at steady state (5 min after the initiation of intravesicular Na(+)-dependent Ca2+ uptake), it was found that the intravesicular Ca2+ content declined with time. Analysis of unidirectional fluxes for Ca2+ at steady state showed that 50 microM XIP inhibited Ca2+ influx and efflux approximately 85 and 70%, respectively. This result suggested that XIP inhibited both Na+/Ca2+ exchange and Ca2+/Ca2+ exchange but had no effect on the passive release pathway for Ca2+. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na+ or other monovalent cations, e.g., K+, is required for XIP to have its inhibitory effect on Ca2+ transport.

Studies of the mechanism underlying increased Na+/Ca2+ exchange activity in Alzheimer's disease brain.

The Na+/Ca2+ exchanger was characterized in plasma membrane vesicles derived from frozen human postmortem tissues. The frontal cortex, temporal cortex and cerebellum of control and Alzheimer's disease (AD) tissues were compared. Na+/Ca2+ exchange activity was defined as the change in vesicular Ca2+ content seen after Na+ loaded vesicles were diluted into choline buffer. The time course of changes in Ca2+ content after dilution was similar in all three regions of control brain. In AD brain, both frontal and temporal cortex vesicles showed elevated Ca2+ content, most evident as an increased peak Ca2+ content at 2 min. The AD cerebellar cortex time course was similar to control and did not show an elevated peak at 2 min. No differences were seen in the passive permeability to Ca2+ when comparing plasma membrane vesicles prepared from control and AD brain. Vesicles from the frontal and temporal cortex of AD brain showed increases in the Vmax of the initial velocity of Ca2+ uptake when compared to control brain, whereas, the cerebellum did not. There were no significant effects of AD on the Km for Ca2+ activation of the initial velocity. Ca2+ influx measured during the rise in vesicular Ca2+ content was elevated in vesicles from AD temporal cortex when compared to control. Two known inhibitors (exchange inhibitory peptide and dichlorobenzamil) of the cardiac Na+/Ca2+ exchanger inhibited the human brain exchanger equally well in control and AD vesicles. Increased Na+/Ca2+ exchange activity was not due to astrocytic gliosis.(ABSTRACT TRUNCATED AT 250 WORDS)

RNA binding by Sxl proteins in vitro and in vivo.

Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.

Analysis of Na+/Ca2+ exchange activity in human brain: the effect of normal aging.

Na+/Ca2+ exchange activity and passive permeability to Ca2+ were analyzed in plasma membrane vesicles (PMV) purified from whole rat brain and three regions of human brain: frontal cortex, temporal cortex, and cerebellum. Accumulation of Ca2+ due to Na+/Ca2+ exchange activity showed a characteristic pattern of an initial rapid rise in Ca2+ content followed by a stable plateau in both rat and human brain. Total Ca2+ accumulation in rat brain PMV was on average three-fold higher than in human brain. Passive permeability to Ca2+ was measured as the rate of Ca2+ release from PMV first loaded with 45Ca by Na+/Ca2+ exchange and then exposed to 1 mM EGTA. The Ca2+ permeabilities of human and rat brain PMV were similar. Ca2+ release from rat brain PMV was faster overall and was resolved into fast and slow components while in human brain a single slow component was found. Post mortem delay up to 4 h had no effect on Na+/Ca2+ exchange Km for Ca2+, Vmax, and peak uptake and Ca2+ release rate in rat brain PMV. Human frontal cortex was shown to have a greater Na+/Ca2+ exchange activity than that found in the cerebellum. The frontal cortex, temporal cortex and cerebellum had similar Ca2+ permeabilities. Age-related effects on Na+/Ca2+ exchange activity and Ca2+ permeability were determined in 15 tissues from human frontal cortex (age at death 21 to 93 years). No significant age related effects were seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural features of the trans-activation response RNA element of equine infectious anemia virus.

A 25-nucleotide RNA with the sequence of the trans-activation response (TAR) element of equine infectious anemia virus (EIAV) was analyzed by biochemical methods and by one- and two-dimensional NMR spectroscopy. NMR, nuclease probing, and polyacrylamide gel migration rates show that the RNA consists of an A-helical stem capped by two non-Watson-Crick U-G base pairs and a compact four-nucleotide loop. The loop is stabilized by base stacking, with loop nucleotides C12 and C15 stacked upon U11 and G16, respectively. Near the 5' end of the molecule, the stem contains a bulge at nucleotide C2, most likely a result of base pairing between G1 and C25. A method for distinguishing RNA stem-loops from palindromic dimers is described and was used to confirm that the EIAV TAR RNA has a stem-loop structure at conditions used for NMR spectroscopy. RNase A and RNase T1 cleavage patterns are consistent with the structural features derived from the NMR data.

Structural features of an RNA containing the CUGGGA loop of the human immunodeficiency virus type 1 trans-activation response element.

A 19-nucleotide RNA containing the CUGGGA loop sequence corresponding to nucleotides 30-35 of the HIV-1 trans-activation response element (TAR) was synthesized in vitro and analyzed by biochemical methods and one- and two-dimensional NMR spectroscopy. Diagnostic RNase cleavage patterns were similar for the loops in the full-length HIV-1 TAR and the 19-nucleotide RNA, indicating that they are similar in structure. NMR data showed that the loop is stabilized by base-stacking interactions. The first loop nucleotide is stacked upon the A-helical stem, and the loop uridine is stacked upon this cytosine. On the opposite side of the loop, the third loop guanosine is stacked upon the adenosine, which is stacked upon the stem. No specific Watson-Crick or non-Watson-Crick base pairing across the loop was identified. Unusually short interribose distances indicate a significant distortion of the sugar-phosphate backbone centered at the adenosine. Relatively short NMR relaxation times for protons of the adenosine and its adjacent guanosine, as well as rapidly exchanging imino protons, provide evidence for dynamic processes occurring in the loop.

Unusual structure of the human immunodeficiency virus type 1 trans-activation response element.

The trans-activation response element (TAR) of human immunodeficiency virus type 1 is a structured RNA consisting of the first 60 nucleotides of all human immunodeficiency virus type 1 RNAs. Computer analyses and limited structural analyses indicated that TAR consists of a stem-bulge-loop structure. Mutational analyses showed that sequences in the bulge are required for Tat binding, whereas sequences in both the bulge and the loop are required for trans activation. In this study, we probed the structures of TAR and various mutants of TAR with chemical probes and RNases and used these methods to footprint a Tat peptide on TAR. Our data show that the structure of wild-type TAR is different from previously published models. The bulge, a Tat-binding site, consists of four nucleotides. The loop is structured, rather than simply single stranded, in a fashion reminiscent of the structures of the tetraloop 5'-UUCG-3' and the GNRA loop (C. Cheong, G. Varani, and I. Tinoco, Jr., Nature [London] 346:680-682, 1990; H.A. Heus and A. Pardi, Science 253:191-193, 1991). RNA footprint data indicate that three bases in the bulge are protected and suggest that a conformational change occurs upon Tat binding.