The importance of being flexible.
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Amino Acid Sequence↗
Biomedical subjects
A D Frankel
Publications and source records attributed to A D Frankel.
Circular dichroism studies suggest that TAR RNA changes conformation upon specific binding of arginine or guanidine.
Short basic peptides from the HIV Tat protein bind specifically to a bulge region in TAR RNA, with a single arginine residue providing the only sequence-specific contact. The free amino acid arginine also binds specifically to TAR. Previous circular dichroism (CD) experiments suggested that peptide binding induces a conformational change in TAR. Here we confirm this observation using single arginine-containing peptides and show that arginine or guanidine binding also induces a conformational change in TAR. A peptide containing a single arginine within a stretch of histidines (CYHHHRHHHHHA) shows pH-dependent binding and a corresponding change in TAR conformation, as detected by a decrease in the CD signal at 265 nm. Arginine and guanidine, which bind to TAR with apparent Kd's of approximately 1.5 mM, induce similar CD changes. In contrast, lysine, which does not bind specifically to TAR, has no effect. Mutants of TAR that abolish specific binding (a U-->C substitution in the three-nucleotide bulge, a deletion of the bulge, or an A-U to U-A base pair change above the bulge) show no change in the CD signal upon binding of peptides, arginine, or guanidine. The results suggest that binding of a single guanidinium group to a specific site in TAR induces a change in RNA conformation.
Amino Acid Sequence↗
Conformation of the TAR RNA-arginine complex by NMR spectroscopy.
The messenger RNAs of human immunodeficiency virus-1 (HIV-1) have an RNA hairpin structure, TAR, at their 5' ends that contains a six-nucleotide loop and a three-nucleotide bulge. The conformations of TAR RNA and of TAR with an arginine analog specifically bound at the binding site for the viral protein, Tat, were characterized by nuclear magnetic resonance (NMR) spectroscopy. Upon arginine binding, the bulge changes conformation, and essential nucleotides for binding, U23 and A27.U38, form a base-triple interaction that stabilizes arginine hydrogen bonding to G26 and phosphates. Specificity in the arginine-TAR interaction appears to be derived largely from the structure of the RNA.
Arginine↗
Specific binding of arginine to TAR RNA.
A single arginine residue within the basic region of the human immunodeficiency virus Tat protein mediates specific binding of Tat peptides to a three-nucleotide bulge in TAR RNA. It has been proposed that arginine recognizes TAR by forming a network of hydrogen bonds with two structurally distinct phosphates, an interaction termed the "arginine fork." Here it is shown that L-arginine blocks the Tat peptide/TAR interaction, whereas L-lysine and analogs of arginine that remove specific hydrogen bond donors do not. Experiments using an L-arginine affinity column demonstrate that arginine and the Tat peptides bind to the same site in TAR. Modification of two phosphates located at the junction of the double-stranded stem and bulge and modification of two adenine N7 groups in base-paired regions of TAR interfere with specific arginine binding. The results emphasize the importance of RNA structure in RNA-protein recognition and provide methods to identify arginine-binding sites in RNAs.
Adenine↗
Specific binding of a basic peptide from HIV-1 Rev.
Human immunodeficiency virus type I (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev activity is mediated through specific binding to a cis-acting Rev responsive element (RRE) located within the env region of HIV-1. A monomer Rev binding site corresponding to 37 nucleotides of the RRE (IIB RNA) was studied by RNA footprinting, modification interference experiments and mutational analysis. Surprisingly, a 17 amino acid peptide, corresponding to the basic domain of Rev, binds specifically to this site at essentially identical nucleotides and probably induces additional base pairing. The Rev protein and related peptide interact primarily with two sets of nucleotides located at the junction of single and double stranded regions, and at an additional site located within a helix. This suggests that the domains of proteins responsible for specific RNA binding can be remarkably small and that the interaction between RNA and protein can probably induce structure in both constituents.
Amino Acid Sequence↗
Peptide models of the Tat-TAR protein-RNA interaction.
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Amino Acid Sequence↗
Activation of HIV transcription by Tat.
Recent studies suggest that the human immunodeficiency virus transactivator, Tat, increases expression of viral genes primarily by enhancing the efficiency of transcriptional elongation. The degree to which Tat influences elongation may depend on the rate of transcriptional initiation. Current models in which Tat interacts with the transcription complex suggest directions for future studies.
Base Sequence↗
Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev.
The Rev protein of HIV-1 regulates the synthesis of partially spliced forms of cytoplasmic viral mRNA by binding to a cis-acting RNA sequence, the Rev response element (RRE). We have investigated the regulation of splicing in vitro and have shown that Rev specifically inhibits splicing of pre-mRNAs containing an RRE by 3- to 4-fold. A synthetic peptide of 17 amino acids containing the RNA-binding domain of Rev is highly functional and specifically inhibits splicing by up to 30-fold. Other peptides that bind to the RRE with high affinity, but with low specificity, do not specifically inhibit splicing. Six repeated monomeric binding sites for the peptide can substitute for the RRE, indicating that regulation by Rev requires interactions with multiple sites. The peptide acts at a step in the assembly of splicing complexes, suggesting that one of the functions of the basic region of Rev is to prevent formation of a functional spliceosome.
Amino Acid Sequence↗
Modular structure of transcription factors: implications for gene regulation.
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DNA-Binding Proteins↗
Arginine-mediated RNA recognition: the arginine fork.
Short peptides that contain the basic region of the HIV-1 Tat protein bind specifically to a bulged region in TAR RNA. A peptide that contained nine arginines (R9) also bound specifically to TAR, and a mutant Tat protein that contained R9 was fully active for transactivation. In contrast, a peptide that contained nine lysines (K9) bound TAR poorly and the corresponding protein gave only marginal activity. By starting with the K9 mutant and replacing lysine residues with arginines, a single arginine was identified that is required for specific binding and transactivation. Ethylation interference experiments suggest that this arginine contacts two adjacent phosphates at the RNA bulge. Model building suggests that the arginine eta nitrogens and the epsilon nitrogen can form specific networks of hydrogen bonds with adjacent pairs of phosphates and that these arrangements are likely to occur near RNA loops and bulges and not within double-stranded A-form RNA. Thus, arginine side chains may be commonly used to recognize specific RNA structures.
Amino Acid Sequence↗
The role of Tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation.
The mechanism of Tat transactivation was studied by treating cell lines containing Tat-defective viruses with purified Tat protein. These cell lines constitutively produce very low levels of virus in the absence of Tat, as measured by p24 antigen levels. Virus production can be increased greater than 30,000-fold by adding exogenous Tat. Tat addition increases mRNA levels early in the viral life cycle, and Tat is required for Rev function to become evident. There is no evidence for a translational effect of Tat. Nuclear run-on experiments show that the increase in mRNA levels is due to an increased efficiency of elongation of nascent transcripts. These results suggest that Tat may be a gene-specific elongation factor.
Amino Acid Sequence↗
Endocytosis and targeting of exogenous HIV-1 Tat protein.
The human immunodeficiency virus-1 (HIV-1) Tat protein has previously been shown to transactivate the HIV-1-LTR when added exogenously to HeLa, H9 lymphocytic and U937 promonocytic cells growing in culture. Here we show that Tat enters these cells by adsorptive endocytosis. Tat appears to bind non-specifically to the cell surface, with greater than 10(7) sites per cell. A specific receptor was not detected by protein crosslinking experiments, and uptake was not affected by treating cells with trypsin, heparinase or neuraminidase. Uptake and transactivation could be inhibited by incubation with heparin, dextran sulfate, an anti-Tat monoclonal antibody, or by incubation at 4 degrees C. In contrast, transactivation by Tat was markedly stimulated by the addition of basic peptides, such as Tat 38-58 or protamine. Fluorescence experiments with rhodamine-conjugated Tat show punctate staining on the cell surface and then localization to the cytoplasm and nucleus. The lack of a specific receptor makes it unclear whether Tat uptake is biologically important in HIV infection, however, the efficiency of uptake raises the possibility that Tat may be useful for delivery of protein molecules into cells.
Amino Acid Sequence↗
Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition.
Arginine-rich sequences are found in many RNA-binding proteins and have been proposed to mediate specific RNA recognition. Fragments of the HIV-1 Tat protein that contain the arginine-rich region of Tat bind specifically to a 3-nucleotide bulge in TAR RNA. To determine the amino acid requirements for specific RNA recognition, we synthesized a series of mutant Tat peptides spanning this domain (YGRKKRRQRRRP) and measured their affinity and specificity for TAR RNA. Several corresponding mutations were introduced into the full-length Tat protein, and trans-activation activity was measured. Systematic substitution of arginine residues with alanines or lysines suggested that overall charge density is important but did not point to any specific residues as being essential for binding. A glutamine-to-alanine substitution had no effect on binding. Remarkably, peptides with scrambled or reversed sequences showed the same affinity and specificity for TAR RNA as the wild-type peptide. Trans-activation activity of the mutant Tat proteins correlated with RNA binding. Arginine-rich peptides from SIV Tat and from HIV-1 Rev, which can functionally substitute for the basic region of HIV-1 Tat, also bound specifically to TAR. Circular dichroism spectra suggest that the arginine-rich region of Tat is unstructured in the absence of RNA, becomes partially or fully structured upon binding, and induces a conformational change in the RNA. These results suggest that arginine-rich RNA-binding domains have considerable sequence flexibility, reminiscent of acidic domains found in transcriptional activators, and that RNA structure may provide much of the specificity for the interaction.
Amino Acid Sequence↗
HIV-1 Tat protein trans-activates transcription in vitro.
Tat protein of human immunodeficiency virus 1 is a potent trans-activator of viral gene expression. We show that purified Tat protein stimulates transcription from viral promoters greater than 10-fold in vitro. A Tat protein mutant that does not trans-activate in vivo did not stimulate transcription in vitro. Tat trans-activation required a functional TAR RNA sequence; trans-activation was competed by the addition of in vitro synthesized wild-type TAR RNA but not by mutant TAR RNAs. That Tat protein directly interacts with the TAR RNA during trans-activation in vitro was suggested by competition with Tat peptides. Preliminary evidence suggests the involvement of a cellular factor in recognition of TAR RNA during Tat trans-activation. Analysis of Tat trans-activation in vitro will provide new mechanistic insights into this process and allow a more detailed study of the relationship between Tat protein structure and function.
Amino Acid Sequence↗
Circular dichroism studies of the HIV-1 Rev protein and its specific RNA binding site.
The circular dichroism (CD) spectrum of the Rev protein from HIV-1 indicates that Rev contains about 50% alpha helix and 25% beta sheet at 5 degrees C in potassium phosphate buffer, pH 3, and 300 mM KF. The spectrum is independent of protein concentration over a 20-fold range. At neutral pH, Rev is relatively insoluble but can be brought into solution by binding to its specific RNA binding site, the Rev-responsive element (RRE), at a Rev:RNA ratio of about 3:1. Nonspecific binding to tRNA does not solubilize Rev. As judged by difference CD spectra, the conformation of Rev when bound to the RRE at neutral pH is similar to the conformation of unbound Rev at pH 3, although changes in the RNA may also contribute to the difference spectrum. Indeed, some difference is observed near 260 nm, consistent with a conformational change of the RRE upon Rev binding. Rev alone at pH 3 shows irreversible aggregation as the temperature is raised, while Rev bound to the RRE at neutral pH shows a reversible transition with a Tm of 68 degrees C.
Amino Acid Sequence↗
Inhibition of antigen-induced lymphocyte proliferation by Tat protein from HIV-1.
The purified human immunodeficiency virus type-l (HIV-l) Tat protein inhibited lymphocyte proliferation induced by tetanus toxoid or Candida antigens by 66 to 97% at nanomolar concentrations of Tat. In contrast, Tat did not cause a significant reduction of lymphocyte proliferation in response to mitogens such as phytohemagglutinin or pokeweed mitogen. Inhibition was blocked by oxidation of the cysteine-rich region of Tat or by incubation with an antibody to Tat before the assay. A synthetic Tat peptide (residues 1 to 58) also inhibited antigen-stimulated proliferation. Experiments with H9 and U937 cell lines showed that Tat can easily enter both lymphocytes and monocytes. The specific inhibition of antigen-induced lymphocyte proliferation by Tat mimics the effect seen with lymphocytes from HIV-infected individuals and suggests that Tat might directly contribute to the immunosuppression associated with HIV infection.
Acquired Immunodeficiency Syndrome↗
Activity of synthetic peptides from the Tat protein of human immunodeficiency virus type 1.
To determine which of the 86 amino acids in the Tat protein of human immunodeficiency virus type 1 (HIV-1) are important for transactivation, peptides from Tat were synthesized and their activity was measured in cells containing a chloramphenicol acetyltransferase reporter gene under control of the HIV long terminal repeat promoter. Although the Tat sequence contains arginine- and cysteine-rich stretches that are difficult to synthesize, it was possible to prepare pure peptides in good yield by using fluoren-9-ylmethoxycarbonyl (Fmoc) chemistry. A peptide containing residues 1-58 had 5-10% the activity of full-length Tat. Deleting 4 amino acids from the N terminus of this peptide further reduced activity, while peptides with more extensive N-terminal deletions and peptides missing the basic region at the C terminus had no detectable activity. A peptide previously reported to transactivate, Tat-(37-62), was completely inactive in our assays. Inactive peptides were also tested as possible inhibitors of transactivation. Tat-(21-38), which contains the cysteine-rich region and can form heterodimers with intact Tat in vitro, showed inhibition at high peptide concentrations. However, this effect was not specific for Tat or for the HIV promoter, since the peptide also inhibited expression from the simian virus 40 early promoter.
Amino Acid Sequence↗