Induced folding in RNA-protein recognition: more than a simple molecular handshake.
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Animals↗
Biomedical subjects
A D Frankel
Publications and source records attributed to A D Frankel.
HIV-1: fifteen proteins and an RNA.
Human immunodeficiency virus type 1 is a complex retrovirus encoding 15 distinct proteins. Substantial progress has been made toward understanding the function of each protein, and three-dimensional structures of many components, including portions of the RNA genome, have been determined. This review describes the function of each component in the context of the viral life cycle: the Gag and Env structural proteins MA (matrix), CA (capsid), NC (nucleocapsid), p6, SU (surface), and TM (transmembrane); the Pol enzymes PR (protease), RT (reverse transcriptase), and IN (integrase); the gene regulatory proteins Tat and Rev; and the accessory proteins Nef, Vif, Vpr, and Vpu. The review highlights recent biochemical and structural studies that help clarify the mechanisms of viral assembly, infection, and replication.
Genes, Viral↗
Molding a peptide into an RNA site by in vivo peptide evolution.
Short peptides corresponding to the arginine-rich domains of several RNA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities. In the case of the HIV-1 Rev-Rev response element (RRE) complex, the peptide forms a single alpha-helix that binds deeply in a widened, distorted RNA major groove and makes a substantial set of base-specific and backbone contacts. Using a reporter system based on antitermination by the bacteriophage lambda N protein, it has been possible to identify novel arginine-rich peptides from combinatorial libraries that recognize the RRE with affinities and specificities similar to Rev but that appear to bind in nonhelical conformations. Here we have used codon-based mutagenesis to evolve one of these peptides, RSG-1, into an even tighter binder. After two rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity and 15-fold higher specificity than the wild-type Rev peptide, and in vitro competition experiments show that RSG-1.2 completely displaces the intact Rev protein from the RRE at low peptide concentrations. By fusing RRE-binding peptides to the activation domain of HIV-1 Tat, we show that the peptides can deliver Tat to the RRE site and activate transcription in mammalian cells, and more importantly, that the fusion proteins can inhibit the activity of Rev in chloramphenicol acetyltransferase reporter assays. The evolved peptides contain proline and glutamic acid mutations near the middle of their sequences and, despite the presence of a proline, show partial alpha-helix formation in the absence of RNA. These directed evolution experiments illustrate how readily complex peptide structures can be evolved within the context of an RNA framework, perhaps reflecting how early protein structures evolved in an "RNA world."
Amino Acid Sequence↗
Dissection of the proposed base triple in human immunodeficiency virus TAR RNA indicates the importance of the Hoogsteen interaction.
A single arginine residue within the RNA-binding domain of the human immunodeficiency virus Tat protein makes a critical sequence-specific contact to TAR RNA. Arginine as the free amino acid also binds specifically to TAR and induces a change in RNA conformation similar to that induced by Tat peptides. NMR and biochemical studies have suggested that the arginine-binding site is stabilized by a base triple interaction between a bulged U and an A x U base pair in the adjacent stem. In this study, we have used chemical modification and mutagenesis experiments to examine the relative contributions of the Watson-Crick and Hoogsteen base-pairing partners of the proposed U-A x U base triple. We show that the Hoogsteen interaction is critical for arginine binding whereas the Watson-Crick interaction can be eliminated or replaced by other base-base interactions. The results are consistent with biochemical studies of the Tat-TAR interaction and support the base triple model for the structure of TAR.
Arginine↗
Alpha helix-RNA major groove recognition in an HIV-1 rev peptide-RRE RNA complex.
The solution structure of a human immunodeficiency virus type-1 (HIV-1) Rev peptide bound to stem-loop IIB of the Rev response element (RRE) RNA was solved by nuclear magnetic resonance spectroscopy. The Rev peptide has an alpha-helical conformation and binds in the major groove of the RNA near a purine-rich internal loop. Several arginine side chains make base-specific contacts, and an asparagine residue contacts a G.A base pair. The phosphate backbone adjacent to a G.G base pair adopts an unusual structure that allows the peptide to access a widened major groove. The structure formed by the two purine-purine base pairs of the RRE creates a distinctive binding pocket that the peptide can use for specific recognition.
Amino Acid Sequence↗
Selection of RNA-binding peptides in vivo.
Many priniciples of sequence-specific DNA recognition have been established over the past decade, largely from structural studies of protein-DNA and drug-DNA complexes. On the basis of these principles, it has been possible to design or select variants of known structural motifs, including zinc-fingers and minor groove-binding drugs, that bind desired sequences. Here we describe a strategy, based on transcriptional termination in bacteria, to identify specific RNA-binding peptides using the arginine-rich RNA-binding motif as a framework. Peptides were isolated from two combinatorial libraries that bind tightly and specifically to the Rev response element of HIV. It appears that alpha-helical peptides resembling Rev were selected from one library whereas new peptides that probably do not form helices were selected from the other, suggesting that the arginine-rich motif may be a particularly versatile framework for recognizing RNA structures.
Amino Acid Sequence↗
Arginine-binding RNAs resembling TAR identified by in vitro selection.
Specific binding of the human immunodeficiency virus Tat protein to its RNA site (TAR) is mediated largely by a single arginine residue located within a basic region of the protein. Many essential features of the interaction can be mimicked by the free amino acid arginine, and an NMR model has been proposed in which the arginine guanidinium group binds to a guanine base in the major groove and to two phosphates adjacent to a bulge, with the RNA structure stabilized by a base triple between a U in the bulge and an adjacent A:U base pair. To compare the TAR structure to other arginine-binding RNAs, we performed in vitro selection experiments and identified RNAs with arginine-binding affinities similar to TAR. About 40% of the selected RNAs contained the same motif found in TAR: two stems separated by a bulge of at least two nucleotides, a U at the 5' position of the bulge, and G:C and A:U base pairs above the bulge. In many cases, the upper stems contained only the G:C and A:U pairs, located next to small loops. Chemical modification experiments demonstrated that these "TAR-like" RNAs bound arginine in a manner similar to TAR, and in some cases identified nucleotides outside the binding site that contributed to binding. To explore how small loops might help stabilize the structures of adjacent arginine-binding sites, we measured arginine-binding affinities of TAR-like RNAs having all possible three-nucleotide loops. An RNA with a UAG loop bound with highest affinity, and chemical modification and RNase mapping experiments suggested that the RNA changes conformation upon arginine binding, converting a large unstructured loop into a bulge conformation related to that of TAR. The results suggest that the arginine-binding site in TAR is structurally versatile and demonstrate how binding can be modulated by the surrounding RNA context.
Arginine↗
Identification of two novel arginine binding DNAs.
RNA tertiary structure is known to play critical roles in RNA-protein recognition and RNA function. To examine how DNA tertiary structure might relate to RNA structure, we performed in vitro selection experiments to identify single-stranded DNAs that specifically bind arginine, and compared the results with analogous experiments performed with RNA. In the case of RNA, a motif related to the arginine binding site in human immunodeficiency virus TAR RNA was commonly found, whereas in the case of DNA, two novel motifs and no TAR-like structures were found. One DNA motif, found in approximately 40% of the cloned sequences, forms of hairpin structure with a highly conserved 10 nucleotide loop, whereas the second motif is especially rich in G residues. Chemical interference and mutagenesis experiments identified nucleotides in both motifs that form specific arginine binding sites, and dimethylsulfate footprinting experiments identified single guanine residues in both that are protected from methylation in the presence of arginine, suggesting possible sites of arginine contact or conformational changes in the DNAs. Circular dichroism experiments indicated that both DNAs undergo conformational changes upon arginine binding and that the arginine guanidinium group alone is responsible for binding. A model for the G-rich motif is proposed in which mixed guanine and adenine quartets may form a novel DNA structure. Arginine binding DNAs and RNAs should provide useful model systems for studying nucleic acid tertiary structure.
Arginine↗
Solution structure of a bovine immunodeficiency virus Tat-TAR peptide-RNA complex.
The Tat protein of bovine immunodeficiency virus (BIV) binds to its target RNA, TAR, and activates transcription. A 14-amino acid arginine-rich peptide corresponding to the RNA-binding domain of BIV Tat binds specifically to BIV TAR, and biochemical and in vivo experiments have identified the amino acids and nucleotides required for binding. The solution structure of the RNA-peptide complex has now been determined by nuclear magnetic resonance spectroscopy. TAR forms a virtually continuous A-form helix with two unstacked bulged nucleotides. The peptide adopts a beta-turn conformation and sits in the major groove of the RNA. Specific contacts are apparent between critical amino acids in the peptide and bases and phosphates in the RNA. The structure is consistent with all biochemical data and demonstrates ways in which proteins can recognize the major groove of RNA.
Amino Acid Sequence↗
Structural variety of arginine-rich RNA-binding peptides.
Arginine-rich domains are used by a variety of RNA-binding proteins to recognize specific RNA hairpins. It has been shown previously that a 17-aa arginine-rich peptide from the human immunodeficiency virus Rev protein binds specifically to its RNA site when the peptide is in an alpha-helical conformation. Here we show that related peptides from splicing factors, viral coat proteins, and bacteriophage antiterminators (the N proteins) also have propensities to form alpha-helices and that the N peptides require helical conformations to bind to their cognate RNAs. In contrast, introducing proline mutations into the arginine-rich domain of the human immunodeficiency virus Tat protein abolishes its potential to form an alpha-helix but does not affect RNA-binding affinity in vitro or in vivo. Based on results from several peptide-RNA model systems, we suggest that helical peptides may be used to recognize RNA structures having particularly wide major grooves, such as those found near loops or large bulges, and that nonhelical or extended peptides may be used to recognize less accessible grooves.
Amino Acid Sequence↗
A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA.
A 17-amino acid arginine-rich peptide from the bovine immunodeficiency virus Tat protein has been shown to bind with high affinity and specificity to bovine immunodeficiency virus transactivation response element (TAR) RNA, making contacts in the RNA major groove near a bulge. We show that, as in other peptide-RNA complexes, arginine and threonine side chains make important contributions to binding but, unexpectedly, that one isoleucine and three glycine residues also are critical. The isoleucine side chain may intercalate into a hydrophobic pocket in the RNA. Glycine residues may allow the peptide to bind deeply within the RNA major groove and may help determine the conformation of the peptide. Similar features have been observed in protein-DNA and drug-DNA complexes in the DNA minor groove, including hydrophobic interactions and binding deep within the groove, suggesting that the major groove of RNA and minor groove of DNA may share some common recognition features.
Amino Acid Sequence↗
Increased cellular uptake of the human immunodeficiency virus-1 Tat protein after modification with biotin.
The human immunodeficiency virus-1 Tat protein can efficiently enter cells when added exogenously in tissue culture. Using the transactivation activity of Tat as a measure of intracellular delivery, we found that the addition of hydrophobic groups to Tat potentiated its uptake. Biotin was the most promising of the reagents tested and we characterized this effect in more detail. When coupled through a cysteine thiol, the addition of a single biotin to Tat increased activity by about six-fold. Increased activity was only seen with reducible biotin analogs, as modification with noncleavable analogs is known to block Tat transactivation activity. Biotin had no effect on Tat uptake when mixed with Tat without cross-linking. Recently, Tat was used as a carrier to direct the uptake of heterologous proteins into cells. We have used RNase as a model system for studying Tat-mediated uptake and found that biotin also increased the delivery of a Tat37-58-RNase conjugate. The increased uptake of Tat and Tat conjugates by addition of hydrophobic groups may significantly enhance the usefulness of Tat as a delivery vehicle, and the approach may be applicable to other systems.
Amino Acid Sequence↗
Assignment and modeling of the Rev Response Element RNA bound to a Rev peptide using 13C-heteronuclear NMR.
The Rev Response Element (RRE) RNA-Rev protein interaction is important for regulation of gene expression in the human immunodeficiency virus. A model system for this interaction, which includes stem IIB of the RRE RNA and an arginine-rich peptide from the RNA-binding domain of Rev, was studied using multidimensional heteronuclear NMR. Assignment of the RNA when bound to the peptide was obtained from NMR experiments utilizing uniformly and specifically 13C-labeled RNA. Isotopic filtering experiments on the specifically labeled RNA enable unambiguous assignment of unusual nonsequential NOE patterns present in the internal loop of the RRE. A three-dimensional model of the RNA in the complex was obtained using restrained molecular dynamics calculations. The internal loop contains two purine-purine base pairs, which are stacked to form one continuous helix flanked by two A-form regions. The formation of a G-G base pair in the internal loop requires an unusual structure of the phosphate backbone. This structural feature is consistent with mutational data as being important for the binding of Rev to the RRE. The G-G base pair may play an important role in opening the normally narrow major groove of A-form RNA to permit binding of the Rev basic domain.
Gene Products, rev↗
Interaction of HIV Rev peptides with the Rev response element RNA.
As a model system for understanding RNA-protein interactions, we are studying the structure of peptides derived from HIV Rev in complex with an RNA derived from the Rev Response Element (RRE) using NMR spectroscopy. Formation of the Rev peptide-RRE complex is accompanied by formation of two purine-purine base pairs in the RRE. These base pairs create an unusual geometry that widens the major groove of the RRE RNA. The Rev peptide is predominantly in an alpha-helical conformation in the complex. The specific recognition of the RRE by Rev involves widening of the major groove to accommodate the Rev alpha-helix.
Base Sequence↗
Costabilization of peptide and RNA structure in an HIV Rev peptide-RRE complex.
An arginine-rich peptide corresponding to amino acids 34-50 of the human immunodeficiency virus Rev protein has been shown to bind specifically to its RNA-binding site (RRE) when the peptide is in an alpha-helical conformation. Mutation of any one of six amino acids (Thr34, Arg35, Arg38, Arg39, Asn40, or Arg44) was shown to strongly decrease specific RNA-binding affinity in vitro, suggesting that these residues may contact specific bases or distinct structural features of the RNA. We now show that the four arginine side chains, and not just their charge, are important for specific binding in vivo, and present evidence that three additional arginines (Arg46, Arg48, and Arg50) may make electrostatic contacts to the RRE. RNA-binding specificity of the Rev peptide is temperature-dependent in vitro, correlating with alpha-helix unfolding. Circular dichroism experiments indicate that the peptide helical structure is stabilized when bound specifically to the RRE and that the RNA undergoes a conformational change upon binding. Because the structures of the peptide and RNA in this model system appear to be mutually stabilized upon binding, it is suggested that the entire complex may be viewed as a single folding unit.
Amino Acid Sequence↗
Binding of an HIV Rev peptide to Rev responsive element RNA induces formation of purine-purine base pairs.
The Rev responsive element (RRE) is an RNA secondary structural element within the env gene of HIV and is the binding site for the viral Rev protein. Formation of the Rev-RRE complex is involved in regulation of splicing and transport of mRNA from the nucleus. To understand the structural basis for the specific recognition of RRE by Rev, we have studied a model system for this interaction using NMR. We have obtained a specific 1:1 complex between an RNA derived from stem IIB of RRE, which contains the highest affinity Rev binding site, and a modified Rev34-50 peptide, which binds the RRE as an alpha-helix [Tan, R., et al. (1993) Cell 73, 1031-1040]. Binding of the peptide was accompanied by a conformational change in the RNA, which resulted in the formation of additional base pairs not present in the free RNA. Two of these induced base pairs are purine-purine pairs within the internal loop of RRE, which had been previously proposed on the basis of biochemical experiments [Bartel, D.P., et al. (1991) Cell 67, 529-536]. The formation of non-Watson-Crick base pairs, interactions in the major groove, and protein-induced conformational changes may prove to be common characteristics of RNA recognition of proteins.
Amino Acid Sequence↗
An RNA-binding peptide from bovine immunodeficiency virus Tat protein recognizes an unusual RNA structure.
The human immunodeficiency virus (HIV) Tat protein binds specifically to an RNA hairpin, TAR, located at the 5' end of its mRNA. Tat uses a single arginine residue within a short region of basic amino acids to recognize a bulge region in TAR. Here we show that a 17 amino acid arginine-rich peptide from the bovine immunodeficiency virus (BIV) Tat protein also binds to an RNA hairpin at the 5' end of its mRNA (BIV TAR), but recognizes different structural features of the RNA. Mutagenesis, RNase mapping, and chemical interference experiments indicate that bulge and stem regions of BIV TAR are recognized simultaneously by the BIV peptide and that the RNA adopts an unusual structure. BIV Tat binds to its TAR site with high affinity and specificity and, unlike HIV Tat, does not appear to use cellular proteins to stabilize RNA binding in vivo. Thus, two related viral activators have evolved rather distinct ways to recognize their RNA targets.
Amino Acid Sequence↗
RNA recognition by an isolated alpha helix.
A 17 amino acid peptide containing the arginine-rich region of the HIV Rev protein binds specifically to Rev response element (RRE) RNA. Even though it is highly charged, the peptide forms an alpha helix in solution, but only when its N- and C-termini are modified to provide favorable electrostatic interactions with the helix macrodipole. Binding affinity for IIB RNA (the primary binding site within the RRE) increases with alpha helix content, whereas nonspecific binding affinity is independent of helix content. Binding of mutant peptides demonstrates that one threonine, one asparagine, and four arginine side chains are important for sequence-specific recognition. Transactivation of the HIV LTR using Tat-Rev peptide hybrids and the RRE IIB site indicates that the peptide adopts an alpha-helical conformation in vivo. The results suggest that interactions with the RNA backbone may help to orient the alpha helix in the major groove of RNA.
Amino Acid Sequence↗