Wild-type TAR Research Articles

Tryptophan residues in TDP-43 and SOD1 modulate the cross-seeding and toxicity of SOD1

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons. Neuronal superoxide dismutase-1 (SOD1) inclusion bodies are characteristic of familial ALS with SOD1 mutations, while a hallmark of sporadic ALS is inclusions containing aggregated WT TAR DNA-binding protein 43 (TDP-43). We show here that co-expression of mutant or WT TDP-43 with SOD1 leads to misfolding of endogenous SOD1 and aggregation of SOD1 reporter protein SOD1G85R-GFP in human cell cultures and promotes synergistic axonopathy in zebrafish. Intriguingly, this pathological interaction is modulated by natively solvent-exposed tryptophans in SOD1 (tryptophan-32) and TDP-43 RNA-recognition motif RRM1 (tryptophan-172), in concert with natively sequestered TDP-43 N-terminal domain tryptophan-68. TDP-43 RRM1 intrabodies reduce WT SOD1 misfolding in human cell cultures, via blocking tryptophan-172. Tryptophan-68 becomes antibody-accessible in aggregated TDP-43 in sporadic ALS motor neurons and cell culture. 5-fluorouridine inhibits TDP-43-induced G85R-GFP SOD1 aggregation in human cell cultures and ameliorates axonopathy in zebrafish, via its interaction with SOD1 tryptophan-32. Collectively, our results establish a novel and potentially druggable tryptophan-mediated mechanism whereby two principal ALS disease effector proteins might directly interact in disease.

Shortening the HIV-1 TAR RNA Bulge by a Single Nucleotide Preserves Motional Modes over a Broad Range of Time Scales.

Helix-junction-helix (HJH) motifs are flexible building blocks of RNA architecture that help define the orientation and dynamics of helical domains. They are also frequently involved in adaptive recognition of proteins and small molecules and in the formation of tertiary contacts. Here, we use a battery of nuclear magnetic resonance techniques to examine how deleting a single bulge residue (C24) from the human immunodeficiency virus type 1 (HIV-1) transactivation response element (TAR) trinucleotide bulge (U23-C24-U25) affects dynamics over a broad range of time scales. Shortening the bulge has an effect on picosecond-to-nanosecond interhelical and local bulge dynamics similar to that casued by increasing the Mg(2+) and Na(+) concentration, whereby a preexisting two-state equilibrium in TAR is shifted away from a bent flexible conformation toward a coaxial conformation, in which all three bulge residues are flipped out and flexible. Surprisingly, the point deletion minimally affects microsecond-to-millisecond conformational exchange directed toward two low-populated and short-lived excited conformational states that form through reshuffling of bases pairs throughout TAR. The mutant does, however, adopt a slightly different excited conformational state on the millisecond time scale, in which U23 is intrahelical, mimicking the expected conformation of residue C24 in the excited conformational state of wild-type TAR. Thus, minor changes in HJH topology preserve motional modes in RNA occurring over the picosecond-to-millisecond time scales but alter the relative populations of the sampled states or cause subtle changes in their conformational features.

Residues at the Cytoplasmic End of Transmembrane Helix 2 Determine the Signal Output of the TarEc Chemoreceptor

Baseline signal output and communication between the periplasmic and cytoplasmic domains of the Escherichia coli aspartate chemoreceptor Tar(Ec) are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). In particular, the cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in wild-type Tar(Ec), is important for determining the CheA kinase-stimulating activity of the receptor and its ability to respond to chemoeffector-induced stimuli. Here, we have studied the effect on Tar(Ec) function of the six-residue sequence at positions 207-212. Moving various combinations of aromatic residues among these positions generates substantial changes in receptor activity. Trp has the largest effect on function, both in maintaining normal activity and in altering activity when it is moved. Tyr has a weaker effect, and Phe has the weakest; however, all three aromatic residues can alter signal output when they are placed in novel positions. We also find that Gly-211 plays an important role in receptor function, perhaps because of the flexibility it introduces into the TM2-HAMP domain connector. The conservation of this Gly residue in the high-abundance chemoreceptors of E. coli and Salmonella enterica suggests that it may be important for the nuanced, bidirectional transmembrane signaling that occurs in these proteins.

Changing the Specificity of a Bacterial Chemoreceptor

The methyl-accepting chemotaxis proteins are a family of receptors in bacteria that mediate chemotaxis to diverse signals. To explore the plasticity of these proteins, we have developed a simple method for selecting cells that swim to target attractants. The procedure is based on establishing a diffusive gradient in semi-soft agar plates and does not require that the attractant be metabolized or degraded. We have applied this method to select for variants of the Escherichia coli aspartate receptor, Tar, that have a new or improved response to different amino acids. We found that Tar can be readily mutated to respond to new chemical signals. However, the overall change in specificity depended on the target compound. A Tar variant that could detect cysteic acid still showed a strong sensitivity to aspartate, indicating that the new receptor had a broadened specificity relative to wild-type Tar. Tar variants that responded to phenylalanine or N-methyl aspartate, or that had an increased sensitivity to glutamate showed a strong decrease in their response to aspartate. In at least some of the cases, the maximal level of sensitivity that was obtained could not be attributed solely to substitutions within the binding pocket. The new tar alleles and the techniques described here provide a new approach for exploring the relationship between ligand binding and signal transduction by chemoreceptors and for engineering new receptors for applications in biotechnology.

Selective binding of TAR RNA by a Tat-derived beta-peptide.

[structure: see text] The interaction between the HIV-1 Tat protein and the TAR RNA element in the nascent viral genomic transcript is required for viral replication. An 11-residue beta-peptide (1), an all-beta homologue of the Arg-rich region Tat 47-57, binds TAR RNA with K(d) = 29 +/- 4 nM. A control beta-peptide (2) in which all Arg side chains are replaced by Lys side chains shows increased affinity but decreased specificity for wild-type vs bulge-deleted TAR RNA, as do the alpha-peptide analogues of 1 and 2.

Intragenic suppressors of a mutation in the aspartate chemoreceptor gene that abolishes binding of the receptor to methyltransferase.

In the chemotaxis of Escherichia coli, receptor methylation is the key process of adaptation. The methyltransferase CheR binds to the carboxy-terminal NWETF sequence of major chemoreceptors. The substitution of Ala for Trp of this sequence (W550A) of the aspartate chemoreceptor (Tar) abolishes its CheR-binding ability. In this study, six independent intragenic suppressors of the mutation were isolated. They were divided into two classes. Tar carrying the class I suppressors (G278A-L488M, T334A, G278A, G278C and A398T) showed signal biases toward tumbling, corresponding to increased activities of the receptor-associated histidine kinase CheA. These suppressors further reduced the unstimulated methylation level of Tar-W550A, but allowed slight but significant stimulation of methylation by aspartate. Some other CheA-activating mutations were also found to serve as class I suppressors. These results suggest that the class I suppressors compensate for the signal bias of Tar-W550A caused by its low methylation level and that the NWETF sequence is required primarily to maintain an appropriate level of methylation by increasing the local concentration of CheR around the receptor. The class II suppressor was a mutation in the termination codon (Op554W) resulting in the addition of 11 residues containing an xWxxF motif. This revertant Tar supported chemotaxis and was methylated almost as effectively as wild-type Tar. This effect was reversed by introducing a mutation in the xWxxF motif. These results reinforce the importance of the xWxxF motif and suggest that the motif does not have to be located at the extreme carboxy terminus.

Collaborative signaling by mixed chemoreceptor teams in Escherichia coli.

Chemoreceptors of the methyl-accepting chemotaxis protein family form clusters, typically at the cell pole(s), in both Bacteria and Archaea. To elucidate the architecture and signaling role of receptor clusters, we investigated interactions between the serine (Tsr) and aspartate (Tar) chemoreceptors in Escherichia coli by constructing Tsr mutations at the six hydrophobic and five polar residues implicated in "trimer of dimers" formation. Tsr mutants with proline replacements could not mediate serine chemotaxis, receptor clustering, or clockwise flagellar rotation. Alanine and tryptophan mutants, although also nonchemotactic, formed receptor clusters, and some produced clockwise flagellar rotation, indicating receptor-coupled activation of the signaling CheA kinase. The alanine and tryptophan mutants evidently assemble defective receptor complexes that cannot modulate CheA activity in response to serine stimuli. In cells containing wild-type Tar receptors, tryptophan replacements in Tsr interfered with Tar function, whereas four Tsr mutants with alanine replacements regained Tsr function. These epistatic and rescuable phenotypes imply interactions between Tsr and Tar dimers in higher-order signaling teams. The bulky side chain in tryptophan mutants may prevent stimulus-induced conformational changes in the team, whereas the small side chain in alanine mutants may permit signaling control when teamed with functional receptor molecules. Direct physical interactions between Tsr and Tar molecules were observed by in vivo chemical crosslinking. Wild-type Tsr crosslinked to Tar, whereas a clustering-defective proline replacement mutant did not. These findings indicate that bacterial chemoreceptor clusters are comprised of signaling teams, seemingly based on trimers of dimers, that can contain different receptor types acting collaboratively.

Conversion of a bacterial warm sensor to a cold sensor by methylation of a single residue in the presence of an attractant.

The aspartate chemoreceptor (Tar) of Escherichia coli also serves as a thermosensor, and it is very amenable to genetic and biochemical analysis of the thermosensing mechanism. Its thermosensing properties are controlled by reversible methylation of the cytoplasmic signalling/adaptation domain of the protein. The unmethylated and the fully methylated (aspartate-bound) receptors sense, as attractant stimuli, increases (warm sensor) and decreases (cold sensor) in temperature respectively. To learn more about the mechanism of thermosensing, we replaced the four methyl-accepting glutamyl residues with non-methylatable aspartyl residues in all possible combinations. In a strain defective in both methyltransferase (CheR) and methylesterase (CheB) activities, all of the mutant Tar proteins functioned as warm sensors. To create a situation in which all of the remaining glutamyl residues were methylated, we expressed the mutant proteins in a CheB-defective, CheR-overproducing strain. The fully glutamyl-methylated proteins were designed to mimic the full range of methylation states possible for wild-type Tar. Almost all of the methylated mutant receptors, including those with single glutamyl residues, were cold sensors in the presence of aspartate. Thus, binding of aspartate to Tar and methylation of its single glutamyl residue can invert its temperature-dependent signalling properties.

Inversion of thermosensing property of the bacterial receptor tar by mutations in the second transmembrane region

The aspartate chemoreceptor Tar of Escherichia coli serves as a warm sensor that produces attractant and repellent signals upon increases and decreases in temperature, respectively. However, increased levels of methylation of the cytoplasmic domain of Tar resulting from aspartate binding convert Tar to a cold sensor with the opposite signaling behavior. Detailed analyses of the methylation sites, which are located in two separate α-helices (MH1 and MH2), have suggested that intra- and/or intersubunit interactions of MH1 and MH2 play a critical role in thermosensing. These interactions may be influenced by binding of aspartate, which could trigger some displacement of MH1 through the second transmembrane region (TM2). As an initial step toward understanding the role of TM2 in thermosensing, we have examined the thermosensing properties of 43 mutant Tar receptors with randomized TM2 sequences (residues 190–210). Among them, we identified one mutant receptor (Tar-I2) that functioned as a cold sensor in the absence of aspartate. This is the first example of attractant-independent inversion of thermosensing in Tar. Further analyses identified the minimal essential divergence from the wild-type Tar sequence (Q191V-W192R-Q193C) required for the inverted response. Thus, displacements of TM2 seem to influence the thermosensing function of Tar.

RNA bulge entropies in the unbound state correlate with peptide binding strengths for HIV-1 and BIV TAR RNA because of improved conformational access.

For the binding of peptides to wild-type HIV-1 and BIV TAR RNA and to mutants with bulges of various sizes, changes in the DeltaDelta G values of binding were determined from experimental K d values. The corresponding entropies of these bulges are estimated by enumerating all possible RNA bulge conformations on a lattice and then applying the Boltzmann relationship. Independent calculations of entropies from fluctuations are also carried out using the Gaussian network model (GNM) recently introduced for analyzing folded structures. Strong correlations are seen between the changes in free energy determined for binding and the two different unbound entropy calculations. The fact that the calculated entropy increase with larger bulge size is correlated with the enhanced experimental binding free energy is unusual. This system exhibits a dependence on the entropy of the unbound form that is opposite to usual binding models. Instead of a large initial entropy being unfavorable since it would be reduced upon binding, here the larger entropies actually favor binding. Several interpretations are possible: (i) the higher conformational freedom implies a higher competence for binding with a minimal strain, by suitable selection amongst the set of already accessible conformations; (ii) larger bulge entropies enhance the probability of the specific favorable conformation of the bound state; (iii) the increased freedom of the larger bulges contri-butes more to the bound state than to the unbound state; (iv) indirectly the large entropy of the bound state might have an unfavorable effect on the solvent structure. Nonetheless, this unusual effect is interesting.

A mechanism for simultaneous sensing of aspartate and maltose by the Tar chemoreceptor of Escherichia coli.

The Tar chemoreceptor of Escherichia coli exhibits partial sensory additivity. Tar can mediate simultaneous responses to two disparate ligands, aspartate and substrate-loaded maltose-binding protein (MBP). To investigate how one receptor generates concurrent signals to two stimuli, ligand-binding asymmetry was imposed on the rotationally symmetric Tar homodimer. Mutations causing specific defects in aspartate or maltose chemotaxis were introduced pairwise into plasmid-borne tar genes. The doubly mutated tar genes did not restore aspartate or maltose chemotaxis in a strain containing a chromosomal deletion of tar (delta tar). However, when Tar proteins with complementing sets of mutations were co-expressed from compatible plasmids, the resulting heterodimeric receptors enabled delta tar cells to respond to aspartate or maltose. The effect of one attractant on the response to the other depended on the relative orientations of the functional binding sites for aspartate and MBP. When the sites were in the 'same' orientation, saturating levels of one attractant strongly inhibited chemotaxis to the other. In the 'opposite' orientation, such inhibitory effects were negligible. These data demonstrate that opposing subunits of Tar can transmit signals to aspartate and maltose independently if the ligands are restricted to the 'opposite' binding orientation. When aspartate and MBP bind in the 'same' orientation, they compete for signalling through one subunit. In the wild-type Tar dimer, aspartate and MBP can bind in either the 'same' or the 'opposite' orientation, a freedom that can explain the partial additivity of the aspartate and maltose responses that is seen with tar+ cells.

Synthesis and RNA polymerase incorporation of the degenerate ribonucleotide analogue rPTP.

The synthesis and enzymatic incorporation into RNA of the hydrogen bond degenerate nucleoside analogue 6-(beta-d-ribofuranosyl)-3, 4-dihydro-8H-pyrimido[4,5-c]-[1,2]oxazin-7-one (P) is described. The 5'-triphosphate of this analogue is readily incorporated by T3, T7 and SP6 RNA polymerases into RNA transcripts, being best incorporated in place of UTP, but also in place of CTP. When all the uridine residues in an HIV-1 TAR RNA transcript are replaced by P the transcript has similar characteristics to the wild-type TAR RNA, as demonstrated by similar melting temperatures and CD spectra. The P-substituted TAR transcript binds to the Tat peptide ADP-1 with only 4-fold lowered efficiency compared with wild-type TAR.

In vitro selection of HIV-1 TAR variants by the Tat protein

Starting from a pool of 101 3 RNA sequences, we isolated a number of TAR RNA variants after nine rounds of selection by binding to recombinant Tat in vitro (SELEX procedure). Sequence analysis of part of the selected molecular species indicated that two TAR variants (clones A and B) were, respectively, represented five and four times. These two groups of sequences constituted approximately 25% of the total number of analyzed clones (9/34). As far as the primary and presumptive secondary structures of the wild-type TAR are concerned, the selected A and B variants showed an almost complete sequence conservation of the Tat-binding domain, but the configuration of this nucleotide region differed within the secondary structure. Despite this difference, as verified by gel retardation and filter binding assays, both the A and B variants bound Tat in vitro with an affinity that was very close to that of the wild-type TAR. Conversely, neither variant sustained Tat-mediated trans-activation in vivo when they replaced the wild-type TAR inside the long terminal repeat of HIV-1. Taken together, our results suggest that these TAR variants have lost the ability to bind cell factor(s) in vivo and may therefore represent useful decoys for the inhibition of HIV-1 replication.

Chemotactic adaptation is altered by changes in the carboxy-terminal sequence conserved among the major methyl-accepting chemoreceptors.

In Escherichia coli and Salmonella typhimurium, methylation and demethylation of receptors are responsible for chemotactic adaptation and are catalyzed by the methyltransferase CheR and the methylesterase CheB, respectively. Among the chemoreceptors of these species, Tsr, Tar, and Tcp have a well-conserved carboxy-terminal motif (NWET/SF) that is absent in Trg and Tap. When they are expressed as sole chemoreceptors, Tsr, Tar, and Tcp support good adaptation, but Trg and Tap are poorly methylated and supported only weak adaptation. It was recently discovered that CheR binds to the NWETF sequence of Tsr in vitro. To examine the physiological significance of this binding, we characterized mutant receptors in which this pentapeptide sequence was altered. C-terminally-mutated Tar and Tcp expressed in a receptorless E. coli strain mediated responses to aspartate and citrate, respectively, but their adaptation abilities were severely impaired. Their expression levels and attractant-sensing abilities were similar to those of the wild-type receptors, but the methylation levels of the mutant receptors increased only slightly upon addition of attractants. When CheR was overproduced, both the adaptation and methylation profiles of the mutant Tar receptor became comparable to those of wild-type Tar. Furthermore, overproduction of CheR also enhanced adaptive methylation of wild-type Trg, which lacks the NWETF sequence, in the absence of any other chemoreceptor. These results suggest that the pentapeptide sequence facilitates effective adaptation and methylation by recruiting CheR.

The single-stranded DNA binding protein, Pur- α, binds HIV-1 TAR RNA and activates HIV-1 transcription

Previous studies indicate that the bulge and loop domains of TAR, the HIV-1 RNA regulatory element, bind viral and cellular factors that are critical for efficient transcription of the HIV-1 genome. In this report, we demonstrate that the cellular protein, Pur- α, a previously characterized sequence specific, single-stranded DNA binding protein, binds to HIV-1 TAR RNA in a specific manner as demonstrated by competition analysis. Pur- α binds to the greatest extent to wild-type TAR RNA, and it appears the primary sequence, as well as the secondary structure and its overall stability contribute to this binding. Results from gel shift analysis using mutant Pur- α proteins indicate that amino acids 55–85, which contain the first of three basic aromatic repeats, are important for its binding to TAR RNA. Overexpression of Pur- α in a glial cell line increased transcription of HIV-1 LTR by a TAR dependent mechanism. The potential contribution by Pur- α to HIV-1 expression in relation to basal transcription by cellular factors is discussed.

TAR- and Tat-independent replication of human immunodeficiency virus type 1 in human hepatoma cells.

The molecular mechanisms involved in the regulation of human immunodeficiency virus type 1 (HIV-1) replication may differ in various cell types and with various exogenous stimuli. TAR/Tat interactions play important roles in HIV-1-long terminal repeat (LTR)-directed transcription, and have become specific targets in molecular therapies for blocking HIV-1 replication. As we previously reported, astrocytic glial cells, which can support HIV-1 replication in cell culture and may be infected in vivo, provide an intracellular milieu in which TAR mutant HIV-1 viruses may replicate. In further studies of this molecular model, several divergent human cell types were analyzed for both TAR- and Tat-independent HIV-1 replication. Human hepatoma cell lines, which can be productively infected by HIV-1 after the hepatoma cells are transduced with the human CD4 receptor gene, were found to support high levels of HIV-1 replication. In these studies, utilizing a transient transfection system with wild-type and various TAR, Tat, or combined TAR/Tat mutant HIV-1 proviral constructs, we demonstrate TAR-independent replication in unstimulated human hepatoma cells. Remarkably, in human hepatoma cells, HIV-1 replication is not only independent of TAR but also can be independent of Tat expression. It is further demonstrated, using electrophoretic mobility shift assays (EMSAs) and an in situ UV cross-linking system, that human hepatoma cells contain novel endogenous cellular proteins that bind to the proviral HIV-1 5' LTR in the downstream region, between nucleotides +38 to +125 on proviral DNA. This alternative regulatory pathway of TAR- and Tat-independent viral production may provide a new system to dissect further the interactions of Tat/TAR and determine the role of the TAR element, in its DNA form, in HIV-1 replication.

Human immunodeficiency virus type 1 TAR element revertant viruses define RNA structures required for efficient viral gene expression and replication.

The TAR element is a viral regulatory element extending from +1 to +60 in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, which is critical for activation by the transactivator protein Tat. Jurkat cell lines chronically infected with viruses containing HIV-1 TAR element mutations are extremely defective for both gene expression and replication. We previously demonstrated that viruses containing mutations of the TAR RNA stem, bulge, or loop structures have 200- to 5,000-fold-reduced levels of gene expression compared with lymphoid cells harboring wild-type virus. In this study, we characterized several Jurkat cell lines infected with TAR element mutant viruses which spontaneously produced culture supernatants with wild-type-like levels of reverse transcriptase activity. These viral supernatants were used to infect Jurkat cells, and following PCR amplification of the viral long terminal repeats, their DNA sequences were analyzed. This analysis demonstrated that revertant viruses isolated from these cell lines retained the original TAR mutations but also contained additional compensatory mutations within TAR. In gel retardation analysis, recombinant Tat protein bound to higher levels to in vitro-transcribed revertant TAR RNAs than the original TAR RNA mutants. Both the original and revertant TAR elements were inserted into both chloramphenicol acetyltransferase reporter and HIV-1 proviral constructs and assayed following transfection of Jurkat cells. Constructs containing revertant TAR element mutations were capable of strong activation by Tat in contrast to constructs containing the original TAR mutations. Analysis of the secondary structure of TAR RNA sequences suggested that TAR RNA structures which differed from that of wild-type TAR were still capable of strong activation in response to Tat. These results further define critical sequences in TAR RNA that are required for tat activation. In addition, since TAR structures with lower free energy that preserve the loop and bulge structures may be favored over fully formed TAR RNA with higher stable free energy, these results implicate nascent RNA rather than the fully formed TAR RNA structure as the target for tat activation.

Interaction of human immunodeficiency virus type 1 Tat-derived peptides with TAR RNA.

Basic peptides from the carboxy terminus of the human immunodeficiency virus type 1 (HIV-1) Tat protein bind to the stem-loop region of TAR RNA, spanning a trinucleotide bulge, with high affinity and moderate specificity. Previous studies have demonstrated that TAR RNA contains a specific arginine binding pocket. A series of 24 amino acid Tat-derived peptides with one or two arginines has been evaluated as possible structural models of the wild-type peptide in its interaction with TAR RNA, using gel electrophoretic methods and circular dichroism (CD) spectroscopy. Dissociation rate measurements indicate that these peptides form complexes with TAR RNA that are significantly less stable kinetically than the wild-type complex. Through a combination of dissociation and association rate measurements, we estimate that wild-type Tat peptide and TAR RNA interact with a Kd of about 16 pM. Together with competition experiments, these results confirm that band shift gel titration methods significantly underestimate absolute peptide-RNA binding affinities in the subnanomolar range. Through competition experiments with bulge mutants of TAR RNA, we demonstrate that peptides that form longer lived complexes with wild-type TAR RNA also show greater discrimination over TAR RNA bulge mutants. Difference CD spectra show that the Tat-derived peptides do not induce the same changes in TAR RNA as the wild-type peptide. The difference CD spectrum of argininamide bound to TAR RNA is most similar to that of the wild-type peptide-TAR RNA complex, implying that the differences in CD spectra upon complex formation are mostly due to changes in TAR RNA conformation.

Requirement for HIV-1 TAR sequences for Tat activation in rodent cells

HIV-1 gene expression is activated via an interaction between the virally encoded Tat protein and a target RNA, TAR.TAR is located at the immediate 5′ end of all viral mRNAs and comprises a partially base-paired stem with a tripyrimidine bulge in the upper stem and a hexanucleotide loop. In vitro, Tat binds specifically to the bulge and upper stem region with no requirement for the loop. In contrast, when Tat activation is analyzed in primate cells, mutations in the loop abolish activation, suggesting a critical role for loop binding cellular factors. However, in rodent cells the reverse is true. Messages with a mutation in the TAR loop are activated whereas messages harboring a wild-type TAR sequence are not activated. By testing the effect of mutations in the bulge and stem in the context of mutation in the loop we now show that this loop-independent activation by Tat in rodent cells requires the critical bulge-stem sequences needed for Tat binding in vitro. These data suggest that in rodent cells, as in vitro, Tat does not require a loop binding cofactor. In rodent cells containing human chromosome 12 (CHO12), however, Tat activation is both bulge and loop dependent. It appears that rodent cells, but not CHO12 cells, are refractory to the normal Tat/TAR activation pathway not by virtue of lacking a loop binding cofactor, but rather by the presence of a loop binding inhibitor of either Tat binding or the activation process.

Hydrogen-bonding Contacts in the Major Groove are required for Human Immunodeficiency Virus Type-1 tat Protein Recognition of TAR RNA

The binding site for tat on TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotides to form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA. Tat binds these duplex RNAs with approximately half the affinity for wild-type TAR RNA. Substitution at positions U23 or U25 by the base analogue. O4-methyl-dT, which is deficient in its ability to hydrogen-bond at the N3 position reduces tat affinity more than 20-fold. Modifications to purines in the stem of TAR RNA that affect hydrogen-bonding ability in either the major or the minor groove of duplex RNA were also tested. Removal of the nitrogen atom at either the N7 position of G26 or the N7 position of A27 reduces tat affinity 10- to 20-fold. By contrast removal of the exocyclic amino group in the minor groove at position G26, by substitution with inosine, does not affect tat binding significantly. A single methylphosphonate substitution at the phosphate bond between A22 and U23 also leads to a significant loss of tat binding ability, whereas all other methylphosphonate substitutions in the U-rich bulge are not harmful to tat binding. We conclude that tat forms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7 of G26, the N7 of A26 and the phosphate between A22 and U23.