ABSTRACT
Site-specific modification of proteins and peptides with reporter molecules provides a powerful research tool in chemistry and biology. We report the synthesis and application of a tyrosine analogue, N-alpha-Fmoc-3-acetyl-L-tyrosine, for selective modification of proteins. As a model system, we synthesized the human immunodeficiency virus type 1 (HIV-1) Tat peptide (amino acids 47-56) containing the arginine rich RNA-binding region and replaced the Tyr-47 with 3-acetyl-tyrosine. The acetyl-Tyr-Tat peptide was subsequently labeled with a fluorescein derivative to study RNA-protein interactions by fluorescence energy transfer experiments. Our results showed that the Tat peptide binds to the rhodamine labeled TAR RNA with a dissociation constant (KD) of 1.0 +/- 0.5 nM. This strategy of selective protein modification offers a versatile new procedure for labeling peptides of biological interest at a desired site when several nucleophilic side chains of lysine and cysteine are present. These methods would provide tools for postsynthetic peptide modification and introducing biophysical probes for structural and functional analysis of proteins.
Subject(s)
Gene Products, tat/chemistry , HIV Long Terminal Repeat , Peptide Fragments/chemistry , Peptide Fragments/chemical synthesis , Protein Engineering/methods , RNA-Binding Proteins/chemistry , Tyrosine/analogs & derivatives , Contrast Media/chemistry , Contrast Media/metabolism , Fluorescein/chemistry , Fluorescein/metabolism , Gene Products, tat/chemical synthesis , Gene Products, tat/genetics , Gene Products, tat/metabolism , Genes, Reporter , Humans , Kinetics , Molecular Structure , Nucleic Acid Conformation , Peptide Fragments/metabolism , Protein Binding , RNA/genetics , RNA/metabolism , RNA-Binding Proteins/chemical synthesis , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. Here we report that two TAR RNA-binding peptidomimetics, oligourea and oligocarbamate, inhibit transcriptional activation by Tat protein in human cells with an IC50 of approximately 0.5 and 1 microM, respectively. Peptidomimetics that can target specific RNA structures provide novel molecules that can be used to control cellular processes involving protein-RNA interactions in vivo.
Subject(s)
HIV-1/drug effects , Polymers/pharmacology , RNA/metabolism , Transcriptional Activation/drug effects , Carbamates/pharmacology , Gene Products, tat/antagonists & inhibitors , HIV-1/genetics , HeLa Cells , Humans , Urea/pharmacology , tat Gene Products, Human Immunodeficiency VirusSubject(s)
Anti-HIV Agents/pharmacology , Virus Replication/drug effects , Amino Acid Sequence , Combinatorial Chemistry Techniques , Gene Products, rev/antagonists & inhibitors , Gene Products, rev/physiology , Gene Products, tat/antagonists & inhibitors , Gene Products, tat/physiology , Molecular Sequence Data , RNA, Viral/metabolismABSTRACT
Human immunodeficiency virus, type 1 (HIV-1), Tat activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex, P-TEFb. Binding of Tat to CycT1 induces cooperative binding of the P-TEFb complex onto nascent HIV-1 TAR RNA. Here the specific interaction between Tat protein, human cyclin T1, and HIV-1 TAR RNA was analyzed by fluorescence resonance energy transfer, using fluorescein-labeled TAR RNA and a rhodamine-labeled Tat protein synthesized through solid-phase chemistry. We find that CycT1 remodels the structure of Tat to enhance its affinity for TAR RNA and that TAR RNA further enhances the interaction between Tat and CycT1. We conclude that TAR RNA nucleates the formation of the Tat.P-TEFb complex through an induced fit mechanism.
Subject(s)
Cyclins/metabolism , Gene Products, tat/metabolism , HIV Long Terminal Repeat/genetics , HIV-1/genetics , RNA, Viral/metabolism , Base Sequence , Cyclin T , Fluorescent Dyes , Humans , Nucleic Acid Conformation , Protein Binding , RNA, Viral/chemistry , Spectrometry, Fluorescence , tat Gene Products, Human Immunodeficiency VirusABSTRACT
Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. A number of cyclic peptides are known to possess antibiotic activity and increased biological stability. Here we report the design, synthesis, and biological activity of a cyclic peptide (2), which inhibits transcriptional activation by Tat protein in human cells with an IC50 of approximately 40 nM. Cyclic peptides that can target specific RNA structures provide a new class of small molecules that can be used to control cellular processes involving RNA-protein interactions in vivo.
Subject(s)
Anti-HIV Agents/chemical synthesis , Gene Products, tat/antagonists & inhibitors , Peptides, Cyclic/chemical synthesis , Anti-HIV Agents/pharmacology , Cells, Cultured , Drug Design , Gene Expression Regulation, Viral/drug effects , Gene Expression Regulation, Viral/genetics , HIV-1/drug effects , HIV-1/genetics , HIV-1/metabolism , HeLa Cells , Humans , Ligands , Peptides, Cyclic/pharmacology , RNA, Viral/drug effects , Transcriptional Activation/drug effects , tat Gene Products, Human Immunodeficiency VirusABSTRACT
Small molecules that bind their biological receptors with high affinity and selectivity can be isolated from randomized pools of combinatorial libraries. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of HIV-1 gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5' end of all nascent HIV-1 transcripts. Here we demonstrate the isolation of small TAR RNA-binding molecules from an encoded combinatorial library. We have made an encoded combinatorial tripeptide library of 24,389 possible members from D-and L-alpha amino acids on TentaGel resin. Using on-bead screening we have identified a small family of mostly heterochiral tripeptides capable of structure-specific binding to the bulge loop of TAR RNA. In vitro binding studies reveal stereospecific discrimination when the best tripeptide ligand is compared with diastereomeric peptide sequences. In addition, the most strongly binding tripeptide was shown to suppress transcriptional activation by Tat protein in human cells with an IC(50) of approximately 50 nM. Our results indicate that tripeptide RNA ligands are cell permeable, nontoxic to cells, and capable of inhibiting expression of specific genes by interfering with RNA-protein interactions.
Subject(s)
Gene Expression Regulation/genetics , RNA/chemistry , Base Sequence , HeLa Cells , Humans , Magnetic Resonance Spectroscopy , Nucleic Acid ConformationABSTRACT
Small unnatural peptides that target specific RNA structures have the potential to control biological processes. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of human immunodeficiency virus type 1 (HIV-1) gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all nascent HIV-1 transcripts. We report here a synthetic peptide derived from Tat sequence (37-72), containing all D-amino acids, that binds in the major groove of TAR RNA and interferes with transcriptional activation by Tat protein in vitro and in HeLa cells. Our results indicate that unnatural peptides can inhibit the transcription of specific genes regulated by RNA-protein interactions.
Subject(s)
Gene Expression Regulation, Viral/drug effects , HIV-1/genetics , Peptides/chemical synthesis , Peptides/pharmacology , Amino Acid Sequence , Binding, Competitive/genetics , Cell-Free System/drug effects , Cell-Free System/metabolism , Cell-Free System/virology , Gene Products, tat/antagonists & inhibitors , Gene Products, tat/chemical synthesis , Gene Products, tat/metabolism , Gene Products, tat/pharmacology , HIV Long Terminal Repeat/drug effects , HeLa Cells , Humans , Molecular Sequence Data , Peptides/antagonists & inhibitors , Peptides/metabolism , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/chemical synthesis , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , RNA, Viral/genetics , Stereoisomerism , Transcriptional Activation/drug effects , tat Gene Products, Human Immunodeficiency VirusABSTRACT
Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans -activation responsive region (TAR) RNA, a 59 base stem-loop structure located at the 5'-end of all HIV transcripts. We have used an intramolecular RNA self-cleaving strategy to determine the folding of TAR RNA and its interactions with a Tat peptide. We incor-porated an EDTA analog at position 24 in the HIV-1 Tat binding site of the TAR RNA. After isolation and purification of the EDTA-TAR conjugate, RNA self-cleavage was initiated by the addition of an iron salt, ascorbate and hydrogen peroxide. Hydroxyl radicals generated from the tethered Fe(II) cleaved TAR RNA backbone in two localized regions. Sites of RNA cleavage were mapped by sequencing reactions. A Tat fragment, Tat(38-72), specifically inhibited RNA self-cleavage. To determine the structural changes caused by the Tat peptide, we performed Fe(II)-EDTA footprinting experiments on Tat-TAR complex. Our high-resolution footprinting results suggest that the inhibition of self-cleavage of EDTA-TAR is due to two effects of Tat binding: (i) Tat binds in the bulge and protects residues in the vicinity of the bulge from self-cleavage and (ii) RNA goes through a structural change where EDTA-U24 is rigidly positioned out of the helix and cannot get access to other nucleotides in the loop of TAR RNA, which are not protected by the Tat peptide. Our results demonstrate that Fe(II)-EDTA-mediated RNA self-cleavage can be applied to study RNA tertiary structures and RNA-protein interactions.