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1.
J Biochem ; 168(2): 139-149, 2020 Aug 01.
Article in English | MEDLINE | ID: mdl-32181810

ABSTRACT

Antizyme (AZ) interacts with ornithine decarboxylase, which catalyzes the first step of polyamine biosynthesis and recruits it to the proteasome for degradation. Synthesizing the functional AZ protein requires transition of the reading frame at the termination codon. This programmed +1 ribosomal frameshifting is induced by polyamines, but the molecular mechanism is still unknown. In this study, we explored the mechanism of polyamine-dependent +1 frameshifting using a human cell-free translation system. Unexpectedly, spermidine induced +1 frameshifting in the mutants replacing the termination codon at the shift site with a sense codon. Truncation experiments showed that +1 frameshifting occurred promiscuously in various positions of the AZ sequence. The probability of this sequence-independent +1 frameshifting increased in proportion to the length of the open reading frame. Furthermore, the +1 frameshifting was induced in some sequences other than the AZ gene in a polyamine-dependent manner. These findings suggest that polyamines have the potential to shift the reading frame in the +1 direction in any sequence. Finally, we showed that the probability of the sequence-independent +1 frameshifting by polyamines is likely inversely correlated with translation efficiency. Based on these results, we propose a model of the molecular mechanism for AZ +1 frameshifting.


Subject(s)
Frameshifting, Ribosomal/genetics , Polyamines/metabolism , Proteins/genetics , HeLa Cells , Humans , Models, Genetic , Proteins/metabolism
2.
Org Biomol Chem ; 17(4): 867-875, 2019 01 23.
Article in English | MEDLINE | ID: mdl-30628612

ABSTRACT

The synthesis of new N1,N8-diacetylspermidine (DiAcSpd) analogues having a linker with desired functional groups in the methylene skeleton, which have been designed by theoretical calculations, is described. We have also achieved the preparation of DiAcSpd supported on solid-phase resins, which have the potential to be used for the evolution of ligands by exponential enrichment (SELEX).

3.
J Biochem ; 161(2): 197-206, 2017 02 01.
Article in English | MEDLINE | ID: mdl-28173167

ABSTRACT

Though polyamines (putrescine, spermidine, and spermine) bind to the specific position in RNA molecules, interaction mechanisms are poorly understood. SELEX procedure has been used to isolate high-affinity oligoribonucleotides (aptamers) from randomized RNA libraries. Selected aptamers are useful in exploring sequences and/or structures in RNAs for binding molecules. In this study, to analyze the interaction mechanism of polyamine to RNA, we selected RNA aptamers targeted for spermine. Two spermine-binding aptamers (#5 and #24) were obtained and both of them had two stem-loop structures. The 3' stem-loop of #5 (SL_2) bound to spermine more effectively than the 5' stem-loop of #5 did. A thermodynamic analysis by an isothermal titration calorimetry revealed that the dissociation constant of SL_2 for spermine was 27.2 µM and binding ratio was nearly 1:1. Binding assay with base-pair replaced variants showed that two stem regions and an internal loop in SL_2 were important for their spermine-binding activities. NMR analyses proposed that a terminal-side and a loop-side stem in SL_2 take a loose and a stable structure, respectively and a conformational change of SL_2 is induced by spermine. It is conclusive that two stems with different characteristics and an internal loop in SL_2 contribute to the specific spermine-binding.


Subject(s)
Aptamers, Nucleotide/chemistry , Spermine/chemistry , Aptamers, Nucleotide/isolation & purification , Binding Sites , Magnetic Resonance Spectroscopy , Nucleic Acid Conformation , Polyamines/chemistry , Thermodynamics
4.
Elife ; 52016 12 23.
Article in English | MEDLINE | ID: mdl-28009256

ABSTRACT

Internal ribosome entry sites (IRESs) are important RNA-based translation initiation signals, critical for infection by many pathogenic viruses. The hepatitis C virus (HCV) IRES is the prototype for the type 3 IRESs and is also invaluable for exploring principles of eukaryotic translation initiation, in general. Current mechanistic models for the type 3 IRESs are useful but they also present paradoxes, including how they can function both with and without eukaryotic initiation factor (eIF) 2. We discovered that eIF1A is necessary for efficient activity where it stabilizes tRNA binding and inspects the codon-anticodon interaction, especially important in the IRES' eIF2-independent mode. These data support a model in which the IRES binds preassembled translation preinitiation complexes and remodels them to generate eukaryotic initiation complexes with bacterial-like features. This model explains previous data, reconciles eIF2-dependent and -independent pathways, and illustrates how RNA structure-based control can respond to changing cellular conditions.


Subject(s)
Eukaryotic Initiation Factor-1/metabolism , Hepacivirus/physiology , Host-Pathogen Interactions , Internal Ribosome Entry Sites , Peptide Chain Initiation, Translational , Ribosomes/metabolism , Viral Proteins/biosynthesis , Cell Line , Humans , Models, Biological
5.
Anal Biochem ; 388(1): 102-7, 2009 May 01.
Article in English | MEDLINE | ID: mdl-19250914

ABSTRACT

Aptamers are short single-stranded DNA or RNA sequences that are selected in vitro based on their high affinity to a target molecule. Here we demonstrate that an RNA aptamer selected against eukaryotic initiation factor 4A (eIF4A) serves as an efficient biosensor. The aptamer, when immobilized to resin, purifies eIF4A from crude cell extracts by affinity pull-down, and 32P-labeled aptamer can detect some 300 ng of eIF4A by dot-blot analysis. Moreover, by use of an aptamer-immobilized sensor chip, we developed a surface plasmon resonance assay to detect eIF4A at the nanogram level within whole cell lysates after optimizing sample preparation, thereby showing a real-time sensor for eIF4A in cell extract solution.


Subject(s)
Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Eukaryotic Initiation Factor-4A/chemistry , Cell Line , DNA, Single-Stranded/chemistry , Eukaryotic Initiation Factor-4A/isolation & purification , Eukaryotic Initiation Factor-4A/metabolism , HeLa Cells , Humans , Immunoblotting
6.
RNA ; 12(10): 1825-34, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16940549

ABSTRACT

Eukaryotic translation initiation factor 4G (eIF4G) plays a crucial multimodulatory role in mRNA translation and decay by interacting with other translation factors and mRNA-associated proteins. In this study, we isolated eight different RNA aptamers with high affinity to mammalian eIF4G by in vitro RNA selection amplification. Of these, three aptamers (apt3, apt4, and apt5) inhibited the cap-dependent translation of two independent mRNAs in a rabbit reticulocyte lysate system. The cap-independent translation directed by an HCV internal ribosome entry site was not affected. Addition of exogenous eIF4G reversed the aptamer-mediated inhibition of translation. Even though apt3 and apt4 were selected independently, they differ only by two nucleotides. The use of truncated eIF4G variants in binding experiments indicated that apt4 (and probably apt3) bind to both the middle and C-terminal domains of eIF4G, while apt5 binds only to the middle domain of eIF4G. Corresponding to the difference in the binding sites in eIF4G, apt4, but not apt5, hindered eIF4G from binding to eIF4A and eIF3, in a purified protein solution system as well as in a crude lysate system. Therefore, the inhibition of translation by apt4 (and apt3) is due to the inhibition of formation of initiation factor complexes involving eIF4A and eIF3. On the other hand, apt5 had a much weaker affinity to eIF4G than apt4, but inhibited translation much more efficiently by an unknown mechanism. The five additional aptamers have sequences and predicted secondary structures that are largely different from each other and from apt3 through apt5. Therefore, we speculate that these seven sets of aptamers may bind to different regions in eIF4G in different fashions.


Subject(s)
Aptamers, Nucleotide/metabolism , Aptamers, Nucleotide/pharmacology , Eukaryotic Initiation Factor-4G/metabolism , Protein Biosynthesis/drug effects , Animals , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/genetics , Base Sequence , Binding Sites , Eukaryotic Initiation Factor-3/metabolism , Eukaryotic Initiation Factor-4A/metabolism , Eukaryotic Initiation Factor-4G/genetics , Eukaryotic Initiation Factors/chemistry , Eukaryotic Initiation Factors/metabolism , Humans , In Vitro Techniques , Molecular Sequence Data , Multiprotein Complexes , Nucleic Acid Conformation , RNA Caps/genetics , RNA Caps/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reticulocytes/metabolism
7.
Nucleic Acids Res ; 33(2): 745-54, 2005.
Article in English | MEDLINE | ID: mdl-15687383

ABSTRACT

A high affinity RNA aptamer (APT58, 58 nt long) against mammalian initiation factor 4A (eIF4A) requires nearly its entire nucleotide sequence for efficient binding. Since splitting either APT58 or eIF4A into two domains diminishes the affinity for each other, it is suggested that multiple interactions or a global interaction between the two molecules accounts for the high affinity. To understand the structural basis of APT58's global recognition of eIF4A, we determined the solution structure of two essential nucleotide loops (AUCGCA and ACAUAGA) within the aptamer using NMR spectroscopy. The AUCGCA loop is stabilized by a U-turn motif and contains a non-canonical A:A base pair (the single hydrogen bond mismatch: Hoogsteen/Sugar-edge). On the other hand, the ACAUAGA loop is stabilized by an AUA tri-nucleotide loop motif and contains the other type of A:A base pair (single hydrogen bond mismatch: Watson-Crick/Watson-Crick). Considering the known structural and functional properties of APT58, we propose that the AUCGCA loop is directly involved in the interaction with eIF4A, while the flexibility of the ACAUAGA loop is important to support this interaction. The Watson-Crick edges of C7 and C9 in the AUCGCA loop may directly interact with eIF4A.


Subject(s)
Antineoplastic Agents/chemistry , Eukaryotic Initiation Factor-4A/antagonists & inhibitors , Models, Molecular , Oligoribonucleotides/chemistry , RNA/chemistry , Antineoplastic Agents/pharmacology , Aptamers, Nucleotide , Base Sequence , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Oligoribonucleotides/pharmacology , RNA/pharmacology
8.
RNA ; 11(1): 77-89, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15611299

ABSTRACT

The eukaryotic translation initiation factor 4F (eIF4F) consists of three polypeptides (eIF4A, eIF4G, and eIF4E) and is responsible for recruiting ribosomes to mRNA. eIF4E recognizes the mRNA 5'-cap structure (m7GpppN) and plays a pivotal role in control of translation initiation, which is the rate-limiting step in translation. Overexpression of eIF4E has a dramatic effect on cell growth and leads to oncogenic transformation. Therefore, an inhibitory agent to eIF4E, if any, might serve as a novel therapeutic against malignancies that are caused by aberrant translational control. Along these lines, we developed two RNA aptamers, aptamer 1 and aptamer 2, with high affinity for mammalian eIF4E by in vitro RNA selection-amplification. Aptamer 1 inhibits the cap binding to eIF4E more efficiently than the cap analog m7GpppN or aptamer 2. Consistently, aptamer 1 inhibits specifically cap-dependent in vitro translation while it does not inhibit cap-independent HCV IRES-directed translation initiation. The interaction between eIF4E and eIF4E-binding protein 1 (4E-BP1), however, was not inhibited by aptamer 1. Aptamer 1 is composed of 86 nucleotides, and the high affinity to eIF4E is affected by deletions at both termini. Moreover, relatively large areas in the aptamer 1 fold are protected by eIF4E as determined by ribonuclease footprinting. These findings indicate that aptamers can achieve high affinity to a specific target protein via global conformational recognition. The genetic mutation and affinity study of variant eIF4E proteins suggests that aptamer 1 binds to eIF4E adjacent to the entrance of the cap-binding slot and blocks the cap-binding pocket, thereby inhibiting translation initiation.


Subject(s)
Eukaryotic Initiation Factor-4E/metabolism , RNA Caps/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Substitution , Animals , Base Sequence , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factors , Genetic Variation , In Vitro Techniques , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Biosynthesis , Protein Conformation , RNA Cap Analogs/chemistry , RNA Cap Analogs/genetics , RNA Cap Analogs/metabolism , RNA Caps/chemistry , RNA Caps/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity
9.
Nucleic Acids Res ; 32(20): 6120-8, 2004.
Article in English | MEDLINE | ID: mdl-15562003

ABSTRACT

The receptor activator of NF-kappaB (RANK) is a member of the tumor necrosis factor (TNF) receptor family and acts to cause osteoclastgenesis through the interaction with its ligand, RANKL. We isolated RNA aptamers with high affinity to human RANK by SELEX. Sequence and mutational analysis revealed that the selected RNAs form a G-quartet conformation that is crucial for binding to RANK. When the aptamer binding to RANK was challenged by RANKL, there was no competition between the aptamer and RANKL. Instead, the formation of a ternary complex, aptamer-RANK-RANKL, was detected by a spin down assay and by BIAcore surface plasmon resonance analysis. Moreover, the selected aptamer efficiently bound to other TNF receptor family proteins, such as TRAIL-R2, CD30, NGFR as well as osteoprotegerin, a decoy receptor for RANK. These results suggest that the selected aptamer recognizes not the ligand-binding site, but rather a common structure conserved in the TNF receptor family proteins.


Subject(s)
Glycoproteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Ribonucleotides/metabolism , Base Sequence , Carrier Proteins/metabolism , Glycoproteins/antagonists & inhibitors , Glycoproteins/chemistry , Humans , Membrane Glycoproteins/metabolism , Nucleic Acid Conformation , Osteoprotegerin , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Tumor Necrosis Factor/chemistry , Ribonucleotides/chemistry , Ribonucleotides/isolation & purification
10.
RNA ; 9(4): 394-407, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12649492

ABSTRACT

The mammalian translation initiation factor 4A (eIF4A) is a prototype member of the DEAD-box RNA helicase family that couples ATPase activity to RNA binding and unwinding. In the crystal form, eIF4A has a distended "dumbbell" structure consisting of two domains, which probably undergo a conformational change, on binding ATP, to form a compact, functional structure via the juxtaposition of the two domains. Moreover, additional conformational changes between two domains may be involved in the ATPase and helicase activity of eIF4A. The molecular basis of these conformational changes, however, is not understood. Here, we generated RNA aptamers with high affinity for eIF4A by in vitro RNA selection-amplification. On binding, the RNAs inhibit ATP hydrolysis. One class of RNAs contains members that exhibit dissociation constant of 27 nM for eIF4A and severely inhibit cap-dependent in vitro translation. The binding affinity was increased on Arg substitution in the conserved motif Ia of eIF4A, which probably improves a predicted arginine network to bind RNA substrates. Selected RNAs, however, failed to bind either domain of eIF4A that had been split at the linker site. These findings suggest that the selected RNAs interact cooperatively with both domains of eIF4A, either in the dumbbell or the compact form, and entrap it into a dead-end conformation, probably by blocking the conformational change of eIF4A. The selected RNAs, therefore, represent a new class of specific inhibitors that are suitable for the analysis of eukaryotic initiation, and which pose a potential therapeutic against malignancies that are caused by aberrant translational control.


Subject(s)
Eukaryotic Initiation Factor-4A/metabolism , Protein Biosynthesis/physiology , RNA/metabolism , Adenosine Triphosphate/metabolism , Base Sequence , Eukaryotic Initiation Factor-4G/metabolism , Molecular Sequence Data , Sequence Analysis, RNA
11.
Biochemistry ; 41(24): 7677-87, 2002 Jun 18.
Article in English | MEDLINE | ID: mdl-12056899

ABSTRACT

The N7-methylguanine portion of the mRNA cap structure interacts with cap-binding proteins via an unusual double-stacking arrangement in which the positively charged cap is sandwiched between two parallel-oriented aromatic protein side chains. Three-dimensional costructures of cap with two mRNA cap-binding proteins, namely, translational initiation factor eIF4E and VP39 (the vaccinia virus-encoded mRNA cap-specific 2'-O-methyltransferase), have heretofore been reported. Despite striking similarities between the two proteins in the double stack with the cap, the stack differs most notably in the species of stacked side chain donated by the protein. Whereas eIF4E employs two tryptophans, VP39 uses a tyrosine and a phenylalanine. Here, we have generated tryptophan substitutions in VP39. Tryptophan substitution was shown, crystallographically, not to disrupt the maintenance of a bona fide parallel stack. However, the single-tryptophan and double-tryptophan substitutions were associated with increased affinity for cap nucleoside by factors of 10 and 50, respectively. VP39 interacted more strongly with a true substrate (containing portions of RNA downstream of the cap in addition to the cap itself) than with isolated cap nucleoside, by several orders of magnitude. VP39 mutants with tryptophan substitution at position 180 exhibited apparent defects in substrate catalytic rate during the first turnover cycle, indicating the possibility of an exquisite sensitivity of the catalytic center to subtle changes in substrate position brought about by alterations in the cap-binding slot. The X-ray structure of VP39 with a genuine nucleobase analogue of N7-methylguanosine, namely, N7,9-dimethylguanine, indicated that the N7-methylguanosine rotational orientation within the stack is a property of the cap nucleobase itself.


Subject(s)
Guanosine/analogs & derivatives , RNA Caps/chemistry , RNA, Messenger/chemistry , RNA-Binding Proteins/chemistry , Vaccinia virus/enzymology , Viral Proteins/chemistry , Crystallography, X-Ray , Eukaryotic Initiation Factor-4E , Guanosine/chemistry , Guanosine/metabolism , Methylation , Methyltransferases/chemistry , Methyltransferases/metabolism , Mutagenesis, Site-Directed , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/metabolism , Protein Binding/genetics , RNA Cap-Binding Proteins , RNA Caps/genetics , RNA Caps/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Solutions , Substrate Specificity/genetics , Vaccinia virus/genetics , Viral Proteins/genetics , Viral Proteins/metabolism
12.
Chem Biol ; 9(6): 679-90, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12079779

ABSTRACT

VP39 is a vaccinia virus-encoded RNA modifying protein with roles in the modification of both mRNA ends. At the 3' end it acts as a processivity factor for the vaccinia poly(A) polymerase (VP55), promoting poly(A) tail elongation. Despite VP39's three-dimensional structure having been elucidated along with details of its mode of mRNA 5' end binding, the VP39-VP55 heterodimer's molecular mechanism of processivity is largely unknown. Here, the area immediately above almost the entire surface of the VP39 subunit was probed using chemical reporters, and the path of a previously unidentified RNA binding site was revealed. The path was indicated to fall within a cleft formed by the intersubunit interface and was consistent with both a previously reported model of the heterodimer-nucleic acid ternary complex and the known function of the heterodimer in processive poly(A) tail elongation.


Subject(s)
Nucleic Acid Conformation , Polynucleotide Adenylyltransferase/metabolism , RNA Caps/metabolism , RNA, Viral/metabolism , Viral Proteins/metabolism , Binding Sites , Dimerization , Ligands , Point Mutation/genetics , Polynucleotide Adenylyltransferase/chemistry , Viral Proteins/chemistry , Viral Proteins/genetics
13.
Microbiology (Reading) ; 146 ( Pt 10): 2595-2603, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11021934

ABSTRACT

Bacillus subtilis FtsY (Srb) is a homologue of the alpha subunit of the receptor for mammalian signal-recognition particle (SRP) and is essential for protein secretion and vegetative cell growth. The ftsY gene is expressed during both the exponential phase and sporulation. In vegetative cells, ftsY is transcribed with two upstream genes, rncS and smc, that are under the control of the major transcription factor sigma(A). During sporulation, Northern hybridization detected ftsY mRNA in wild-type cells, but not in sporulating cells of sigma(K) and gerE mutants. Therefore, ftsY is solely expressed during sporulation from a sigma(K)- and GerE-controlled promoter that is located immediately upstream of ftsY inside the smc gene. To examine the role of FtsY during sporulation, the B. subtilis strain ISR39 was constructed, a ftsY conditional mutant in which ftsY expression can be shut off during spore formation but not during the vegetative state. Electron microscopy showed that the outer coat of ISR39 spores was not completely assembled and immunoelectron microscopy localized FtsY to the inner and outer coats of wild-type spores.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Sigma Factor , Bacillus subtilis/metabolism , Bacillus subtilis/physiology , Base Sequence , Gene Deletion , Microscopy, Electron/methods , Microscopy, Immunoelectron/methods , Molecular Sequence Data , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Peptide/chemistry , Restriction Mapping , Spores, Bacterial/genetics , Spores, Bacterial/physiology , Transcription Factors
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