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1.
Kohei Yamashita Takahiro Tanaka Hiroyuki Furuta Yoshiya Ikawa 《Journal of peptide science》2012,18(10):635-642
TectoRNA, an artificial RNA with self‐assembling ability, has been employed as a structural platform for RNA nanotechnology and RNA synthetic biology. In this study, tectoRNA was applied as a specific template for chemical peptide ligation. On the basis of a self‐assembling tectoRNA, we designed and constructed a template RNA that facilitates peptide ligation depending on controlled dimer formation. Two RNA‐binding peptides were recognized by two peptide‐binding RNA motifs embedded in the template RNA, and chemical ligation was promoted because of the entropic effect of Mg2+‐dependent dimerization. In a series of biochemical analyses, we determined the relationship between the structures of the tectoRNA‐based templates and the extent of acceleration in peptide ligation. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
2.
Yamashita K Kashiwagi N Furuta H Ikawa Y 《Bioscience, biotechnology, and biochemistry》2011,75(10):2021-2024
A designed self-folding RNA possessing two peptide-recognition motifs served as a template for the chemical ligation of two RNA-binding peptides under stoichiometric conditions. In this study, we investigated the turnover ability of this template RNA in facilitation of peptide ligation and found that the RNA exhibited modest turnover ability under conditions in which its 3D structure was marginally stable. 相似文献
3.
Koo BK Park CJ Fernandez CF Chim N Ding Y Chanfreau G Feigon J 《Journal of molecular biology》2011,411(5):927-2421
H/ACA small nucleolar and Cajal body ribonucleoproteins (RNPs) function in site-specific pseudouridylation of eukaryotic rRNA and snRNA, rRNA processing, and vertebrate telomerase biogenesis. Nhp2, one of four essential protein components of eukaryotic H/ACA RNPs, forms a core trimer with the pseudouridylase Cbf5 and Nop10 that binds to H/ACA RNAs specifically. Crystal structures of archaeal H/ACA RNPs have revealed how the protein components interact with each other and with the H/ACA RNA. However, in place of Nhp2p, archaeal H/ACA RNPs contain L7Ae, which binds specifically to an RNA K-loop motif absent from eukaryotic H/ACA RNPs, while Nhp2 binds a broader range of RNA structures. We report solution NMR studies of Saccharomyces cerevisiae Nhp2 (Nhp2p), which reveal that Nhp2p exhibits two major conformations in solution due to cis/trans isomerization of the evolutionarily conserved Pro83. The equivalent proline is in the cis conformation in all reported structures of L7Ae and other homologous proteins. Nhp2p has the expected α-β-α fold, but the solution structures of the major conformation of Nhp2p with trans Pro83 and of Nhp2p-S82W with cis Pro83 reveal that Pro83 cis/trans isomerization affects the positions of numerous residues at the Nop10 and RNA binding interface. An S82W substitution, which stabilizes the cis conformation, also stabilizes the association of Nhp2p with H/ACA snoRNPs expressed in vivo. We propose that Pro83 plays a key role in the assembly of the eukaryotic H/ACA RNP, with the cis conformation locking in a stable Cbf5-Nop10-Nhp2 ternary complex and positioning the protein backbone to interact with the H/ACA RNA. 相似文献
4.
Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules
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Gallois-Montbrun S Kramer B Swanson CM Byers H Lynham S Ward M Malim MH 《Journal of virology》2007,81(5):2165-2178
Members of the APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1-like) family of cytidine deaminases inhibit host cell genome invasion by exogenous retroviruses and endogenous retrotransposons. Because these enzymes can edit DNA or RNA and potentially mutate cellular targets, their activities are presumably regulated; for instance, APOBEC3G (A3G) recruitment into high-molecular-weight ribonucleoprotein (RNP) complexes has been shown to suppress its enzymatic activity. We used tandem affinity purification together with mass spectrometry (MS) to identify protein components within A3G-containing RNPs. We report that numerous cellular RNA-binding proteins with diverse roles in RNA function, metabolism, and fate determination are present in A3G RNPs but that most interactions with A3G are mediated via binding to shared RNAs. Confocal microscopy demonstrated that substantial quantities of A3G localize to cytoplasmic microdomains that are known as P bodies and stress granules (SGs) and are established sites of RNA storage and metabolism. Indeed, subjecting cells to stress induces the rapid redistribution of A3G and a number of P-body proteins to SGs. Among these proteins are Argonaute 1 (Ago1) and Argonaute 2 (Ago2), factors that are important for RNA silencing and whose interactions with A3G are resistant to RNase treatment. Together, these findings reveal that A3G associates with RNPs that are found throughout the cytosol as well as in discrete microdomains. We also speculate that the interplay between A3G, RNA-silencing pathways, and cellular sites of RNA metabolism may contribute to A3G's role as an inhibitor of retroelement mobility and as a possible regulator of cellular RNA function. 相似文献
5.
Putative intermediary stages for the molecular evolution from a ribozyme to a catalytic RNP 总被引:1,自引:1,他引:0
A hypothetical evolutionary pathway from a ribozyme to a catalytic RNA–protein complex (RNP) is proposed and examined. In this hypothesis for an early phase of molecular evolution, one RNA–RNA interaction in the starting ribozyme is replaced with an RNA–protein interaction via two intermediary stages. At each stage, the original RNA–RNA interaction and a newly introduced RNA–protein interaction are designed to coexist. The catalytic RNPs corresponding to the intermediary stages were constructed by employing the Tetrahymena ribozyme together with molecular modeling. Analyses of the RNPs indicate that the protein can fully replace the original role of the RNA–RNA interaction in the starting ribozyme and that the association of a protein with a ribozyme might be beneficial for improving the ribozymatic activity. 相似文献
6.
Abstract
Short oligonucleotide and peptide replicators have been described. To determine whether cross-replication could have occurred
between such systems, we have attempted to show that peptides can specifically template the ligation of nucleic acids. A complex
between a 35-mer anti-Rev RNA aptamer and a 17-mer arginine-rich motif (ARM) peptide from the HIV-1 Rev protein served as
a model system. Aptamer half-molecules were activated for ligation via two activation chemistries, representing two distinct
kinetic possibilities for early replicators. Cyanogen bromide activation was transient relative to oligonucleotides that terminated
with a 5′-iodine and a 3′phosphorothioate, respectively. The Rev ARM specifically enhanced the degree or rate of ligation
by both methods: there was a 10-fold increase in the production of full-length aptamer in the presence of cyanogen bromide
and a 5.9- to 7.6-fold enhancement in the rate of ligation for stably activated aptamer half-molecules. These results support
the possibility that life could have originated with peptide replicators and transitioned to nucleic acid replicators or that
peptide and nucleic acid replicators could have been interdependent. 相似文献
7.
Anna Lena Gell Nadja Groysbeck Christian F. W. Becker Anne C. Conibear 《Journal of peptide science》2017,23(12):871-879
A prominent target of monoclonal antibodies as targeted therapies for cancer is the epidermal growth factor receptor, which is overexpressed on the surface of various cancer cell types. Its natural binder, the epidermal growth factor (EGF), is a 53 amino acid polypeptide. Anticancer synthetic targeted immune system engagers (ISErs) comprising two ‘binder’ peptides, which are attached to a scaffold conveying immune stimulating ‘effector’ properties, via monodisperse polyethylene glycol chains. So far, preparation of ISErs has been limited to the use of small peptides (8–20 amino acids) as binding functionalities, and they have been entirely synthesized by solid phase peptide synthesis. Here, we describe a synthetic and a semisynthetic approach for the preparation of an ISEr bearing two murine EGF molecules as binding entities (ISEr‐EGF2). EGF was either synthesized in segments by solid phase peptide synthesis or expressed recombinantly and ligated to the scaffold by native chemical ligation. We report the successful generation of synthetic and semisynthetic ISEr‐EGF2 as well as several challenges encountered during the synthesis and ligations. We demonstrate the application of native chemical ligation for the design of larger ISEr constructs, facilitating new objectives for the coupling of small binder peptides and larger proteins to multivalent ISEr scaffolds. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
8.
Background
Ro ribonucleoprotein particles (Ro RNPs) consist of a non-coding Y RNA bound by Ro60, La and possibly other proteins. The physiological function of Ro RNPs is controversial as divergent functions have been reported for its different constituents. We have recently shown that Y RNAs are essential for the initiation of mammalian chromosomal DNA replication, whereas Ro RNPs are implicated in RNA stability and RNA quality control. Therefore, we investigate here the functional consequences of RNP formation between Ro60, La and nucleolin proteins with hY RNAs for human chromosomal DNA replication.Methodology/Principal Findings
We first immunoprecipitated Ro60, La and nucleolin together with associated hY RNAs from HeLa cytosolic cell extract, and analysed the protein and RNA compositions of these precipitated RNPs by Western blotting and quantitative RT-PCR. We found that Y RNAs exist in several RNP complexes. One RNP comprises Ro60, La and hY RNA, and a different RNP comprises nucleolin and hY RNA. In addition about 50% of the Y RNAs in the extract are present outside of these two RNPs. Next, we immunodepleted these RNP complexes from the cytosolic extract and tested the ability of the depleted extracts to reconstitute DNA replication in a human cell-free system. We found that depletion of these RNP complexes from the cytosolic extract does not inhibit DNA replication in vitro. Finally, we tested if an excess of recombinant pure Ro or La protein inhibits Y RNA-dependent DNA replication in this cell-free system. We found that Ro60 and La proteins do not inhibit DNA replication in vitro.Conclusions/Significance
We conclude that RNPs containing hY RNAs and Ro60, La or nucleolin are not required for the function of hY RNAs in chromosomal DNA replication in a human cell-free system, which can be mediated by Y RNAs outside of these RNPs. These data suggest that Y RNAs can support different cellular functions depending on associated proteins. 相似文献9.
In Drosophila, neurosecretory cells that release peptide hormones play a prominent role in the regulation of development, growth, metabolism, and reproduction. Several types of peptidergic neurosecretory cells have been identified in the brain of Drosophila with release sites in the corpora cardiaca and anterior aorta. We show here that in adult flies the products of three neuropeptide precursors are colocalized in five pairs of large protocerebral neurosecretory cells in two clusters (designated ipc-1 and ipc-2a): Drosophila tachykinin (DTK), short neuropeptide F (sNPF) and ion transport peptide (ITP). These peptides were detected by immunocytochemistry in combination with GFP expression driven by the enhancer trap Gal4 lines c929 and Kurs-6, both of which are expressed in ipc-1 and 2a cells. This mix of colocalized peptides with seemingly unrelated functions is intriguing and prompted us to initiate analysis of the function of the ten neurosecretory cells. We investigated the role of peptide signaling from large ipc-1 and 2a cells in stress responses by monitoring the effect of starvation and desiccation in flies with levels of DTK or sNPF diminished by RNA interference. Using the Gal4-UAS system we targeted the peptide knockdown specifically to ipc-1 and 2a cells with the c929 and Kurs-6 drivers. Flies with reduced DTK or sNPF levels in these cells displayed decreased survival time at desiccation and starvation, as well as increased water loss at desiccation. Our data suggest that homeostasis during metabolic stress requires intact peptide signaling by ipc-1 and 2a neurosecretory cells. 相似文献
10.
Unusual features of fibrillarin cDNA and gene structure in Euglena gracilis: evolutionary conservation of core proteins and structural predictions for methylation-guide box C/D snoRNPs throughout the domain Eucarya
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Box C/D ribonucleoprotein (RNP) particles mediate O2′-methylation of rRNA and other cellular RNA species. In higher eukaryotic taxa, these RNPs are more complex than their archaeal counterparts, containing four core protein components (Snu13p, Nop56p, Nop58p and fibrillarin) compared with three in Archaea. This increase in complexity raises questions about the evolutionary emergence of the eukaryote-specific proteins and structural conservation in these RNPs throughout the eukaryotic domain. In protists, the primarily unicellular organisms comprising the bulk of eukaryotic diversity, the protein composition of box C/D RNPs has not yet been extensively explored. This study describes the complete gene, cDNA and protein sequences of the fibrillarin homolog from the protozoon Euglena gracilis, the first such information to be obtained for a nucleolus-localized protein in this organism. The E.gracilis fibrillarin gene contains a mixture of intron types exhibiting markedly different sizes. In contrast to most other E.gracilis mRNAs characterized to date, the fibrillarin mRNA lacks a spliced leader (SL) sequence. The predicted fibrillarin protein sequence itself is unusual in that it contains a glycine-lysine (GK)-rich domain at its N-terminus rather than the glycine-arginine-rich (GAR) domain found in most other eukaryotic fibrillarins. In an evolutionarily diverse collection of protists that includes E.gracilis, we have also identified putative homologs of the other core protein components of box C/D RNPs, thereby providing evidence that the protein composition seen in the higher eukaryotic complexes was established very early in eukaryotic cell evolution. 相似文献
11.
Lauren A. Michael 《Journal of molecular biology》2009,392(3):774-715
Recognition of the human immunodeficiency virus Rev-responsive element (RRE) RNA by the Rev protein is an essential step in the viral life cycle. Formation of the Rev-RRE complex signals nucleocytoplasmic export of unspliced and partially spliced viral RNA. Essential components of the complex have been localized to a minimal arginine-rich Rev peptide and stem IIB of RRE. In vitro selection studies have identified a synthetic peptide known as RSG 1.2 that binds with better specificity and affinity to RRE than the Rev peptide. NMR structures of both peptide-RNA complexes of Rev and RSG 1.2 bound to RRE stem IIB have been solved and reveal gross structural differences between the two bound complexes. Molecular dynamics simulations of the Rev and RSG 1.2 peptides in complex with RRE stem IIB have been simulated to better understand on an atomic level how two arginine-rich peptides of similar length recognize the same sequence of RNA with such different structural motifs. While the Rev peptide employs some base-specific hydrogen bonding for recognition of RRE, shape recognition, through contact with the sugar-phosphate backbone, and cation-pi interactions are also important. Molecular dynamics simulations suggest that RSG 1.2 binds more tightly to the RRE sequence than Rev by forming more base-specific contacts, using water to mediate peptide-RNA contacts, and is held in place by a strong salt bridge network spanning the major groove of the RNA. 相似文献
12.
《Peptides》2012,33(12):2418-2427
Ubiquitin is a conservative polypeptide present in every eukaryotic cell. Apart from its involvement in proteasomal degradation and other intracellular signal pathways, it was suggested to play an important role as the extracellular immunomodulator and antimicrobial agent. Moreover, ubiquitin-derived peptides were shown to express significant biological activities. Our previous studies showed a high immunosuppressive potency of the ubiquitin peptic hydrolysate in which we identified over 70 different peptides. The present work focuses on synthesizing the most abundant of these peptides and investigating their immunomodulatory potency. The peptide VKTLTGKTI possessed the highest immunosuppressory activity in AFC experiments, comparable to the previously described LEDGRTLSDY sequence (a previously discovered ubiquitin-derived peptide). Moreover, some of the investigated peptides expressed immunostimulatory effects. These findings support the idea that ubiquitin, together with products of its degradation, could represent a self-regulating immunoregulatory system. Peptide VKTLTGKTI was also tested for its activity to prolong the skin graft survival in mice. The results showed that the investigated peptide significantly extended the skin transplant rejection time, therefore it could be considered as a potential supplementary medicine in the post-transplantation therapy. Moreover, we synthesized two analogs of investigated peptides, first designed to mimic the non-linear epitope consisting of ubiquitin 16–21 and ubiquitin 52–57 fragments, and second designed to mimic the ubiquitin 5–13 hairpin. We also tested their immunosuppressory activity in in vitro experiments. 相似文献
13.
《Peptides》2016
A small library of amphiphilic peptides has been evaluated for duplex RNA carrier function into A549 cells. We studied peptides in which a C-terminal 7-residue cationic domain is attached to a neutral/hydrophobic 23-residue domain that is based on the viral fusion peptide of HIV. We also examined peptides in which the cationic charge was evenly distributed throughout the peptide. Strikingly, subtle sequence variations in the hydrophobic domain that do not alter net hydrophobicity result in wide variation in RNA uptake. Additionally, cyclic cystine variants are much less active as RNA carriers than their open-chain cysteine analogs. With regard to electrostatic effects, we find that lysine is less effective than arginine in facilitating uptake, and that even distribution of cationic residues throughout the peptide sequence results in especially effective RNA carrier function. Overall, minor changes in peptide hydrophobicity, flexibility and charge distribution can significantly alter carrier function. We hypothesize this is due to altered properties of the peptide-RNA assembly rather than peptide secondary structure. 相似文献
14.
《Journal of molecular biology》1996,258(1):53-61
Antibiotics act as inhibitors of various biological processes. Here we demonstrate that some tuberactinomycins, hitherto known as inhibitors of prokaryotic protein synthesis and of group I intron self-splicing, have a modulatory effect on group I intron RNAs. The linear intron, which is excised during the self-splicing process, is still an active molecule capable of performing an intramolecular transesterification resulting in a circular molecule. However, in the presence of sub-inhibitory concentrations of tuberactinomycins, the intron reacts intermolecularly leading to the formation of linear head-to-tail intron-oligomers. The antibiotic stimulates the intron to reactin transinstead ofin cis. The phage T4-derivedtdintron uses the same sites for oligomerisation as for circularisation. Gel-retardation experiments demonstrate that the intron RNA forms non-covalent complexes in the presence of the antibiotic. It might be envisaged that the role of these peptide antibiotics is to bridge RNA molecules mediating RNA – RNA interactions and thus enabling their reaction. The tuberactinomycins are further able to induce the interaction of heterologous introns. The ligation of the T4 phage-derivedtdintron with theTetrahymenarRNA intron is very efficient, resulting in molecules composed of two introns derived from different species. Thetdintron attacks theTetraymenaintron at various sites, which are located within double-stranded regions. These observations suggest that small molecules like these basic peptide antibiotics could have mediated RNA–RNA interactions in a pre-protein era. 相似文献
15.
Alu elements are the most abundant repetitive elements in the human genome; they emerged from the signal recognition particle RNA gene and are composed of two related but distinct monomers (left and right arms). Alu RNAs transcribed from these elements are present at low levels at normal cell growth but various stress conditions increase their abundance. Alu RNAs are known to bind the cognate proteins SRP9/14. We purified synthetic Alu RNP, composed of Alu RNA in complex with SRP9/14, and investigated the effects of Alu RNPs and naked Alu RNA on protein translation. We found that the dimeric Alu RNP and the monomeric left and right Alu RNPs have a general dose-dependent inhibitory effect on protein translation. In the absence of SRP9/14, Alu RNA has a stimulatory effect on all reporter mRNAs. The unstable structure of sRight RNA suggests that the differential activities of Alu RNP and Alu RNA may be explained by conformational changes in the RNA. We demonstrate that Alu RNPs and Alu RNAs do not stably associate with ribosomes during translation and, based on the analysis of polysome profiles and synchronized translation, we show that Alu RNP and Alu RNA regulate translation at the level of initiation. 相似文献
16.
Maki Sugaya Ryota Saito Yuriko Matsumura Kazuo Harada Akira Katoh 《Journal of peptide science》2008,14(8):978-983
A simple method for the detection of specific RNA-polypeptide interactions using MALDI-TOF mass spectroscopy is described. Instead of direct observation of the RNA-polypeptide complex, we attempted the indirect observation of the binding event by focusing on the disappearance of the free polypeptide signal upon interaction with RNA. As a result, specific binding of the Rev-response element (RRE) RNA of the HIV with two RRE-binding peptide aptamers, DLA and RLA peptides, as well as the bacteriophage lambda boxB RNA with the lambda N peptide was observed. We also show that specific RNA-binding peptides can be identified from a mixture of peptides with varying RNA-binding affinity, showing that the method could be applied to high-throughput screening from simple peptide libraries. The method described in this study provides a quick and simple method for detecting specific RNA-polypeptide interactions that avoids difficulties associated with direct observation of RNA and RNA-polypeptide complexes, which may find various applications in the analysis of RNA-polypeptide interactions and in the identification of novel RNA-binding polypeptides. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
17.
Effects on translation pausing of alterations in protein and RNA components of the ribosome exit tunnel 总被引:1,自引:0,他引:1
Amino acids are polymerized into peptides in the peptidyl transferase center of the ribosome. The nascent peptides then pass through the exit tunnel before they reach the extraribosomal environment. A number of nascent peptides interact with the exit tunnel and stall elongation at specific sites within their peptide chain. Several mutational changes in RNA and protein components of the ribosome have previously been shown to interfere with pausing. These changes are localized in the narrowest region of the tunnel, near a constriction formed by ribosomal proteins L4 and L22. To expand our knowledge about peptide-induced pausing, we performed a comparative study of pausing induced by two peptides, SecM and a short peptide, CrbCmlA, that requires chloramphenicol as a coinducer of pausing. We analyzed the effects of 15 mutational changes in L4 and L22, as well as the effects of methylating nucleotide A2058 of 23S rRNA, a nucleotide previously implicated in pausing and located close to the L4-L22 constriction. Our results show that methylation of A2058 and most mutational changes in L4 and L22 have differential effects on pausing in response to CrbCmlA and SecM. Only one change, a 6-amino-acid insertion after amino acid 72 in L4, affects pausing in both peptides. We conclude that the two peptides interact with different regions of the exit tunnel. Our results suggest that either the two peptides use different mechanisms of pausing or they interact differently but induce similar inhibitory conformational changes in functionally important regions of the ribosome. 相似文献
18.
Maki Sugaya Norikazu Nishino Akira Katoh Kazuo Harada 《Journal of peptide science》2008,14(8):924-935
The arginine-rich motif is a class of short arginine-rich peptides that bind to specific RNA structures that has been found to be a versatile framework for the design and selection of RNA-binding peptides. We previously identified novel peptides that bind to the Rev-response element (RRE) RNA of the HIV from an arginine-rich polypeptide library (ARPL) consisting of a polyarginine (15 mer) randomized at the N-terminal 10 positions. The selected peptides bound more strongly to the RRE than the natural binding partner, Rev, and contained glutamine residues that were assumed to be important for recognition of the G-A base pair. In addition, the peptides were predicted to bind to the RRE in an alpha-helical conformation. In this study, in order to understand the mechanism of the interaction between the RRE and the putative alpha-helical glutamine-containing peptides, the amino acid requirements for high affinity binding were analyzed by a combinatorial approach using a bacterial system for detecting RNA-peptide interactions. A consensus peptide, the DLA peptide, was elucidated, which consists of a single glutamine residue within a polyarginine context with the glutamine residue flanked at specific positions by three nonarginine residues, two of which appear to be important for alpha-helix stabilization. In addition, the DLA peptide was found to bind extremely tightly to the RRE with an affinity 50-fold higher than that of the Rev peptide as determined by a gel shift assay. A working model for the interaction of the DLA peptide to the RRE is proposed, which should aid in the development of peptide-based drugs that inhibit HIV replication, as well as in our understanding of polypeptide-RNA interactions. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
19.
The four alfalfa mosaic virus RNAs (respectively 24 S, 20 S, 17 S and 12 S) have been used separately as messengers in two in vitro protein synthesizing systems: wheat germ and rabbit reticulocyte lysate. In both systems a polypeptide corresponding to the translation of the entire length of the RNA can be found for RNAs 24 S, 20 S and 12 S, but not for 17 S RNA, the translation product of which is only 35,000 daltons. The number of initiation sites has been determined for each RNA by analyzing the initiation peptides synthesized in the presence of spasomycin and show that there is only one initiation or binding site perRNA. We thus conclude that each AMV RNA behaves as a monocistronic messenger in in vitro translating systems. 相似文献
20.
D. W. Kingsbury 《Journal of virology》1973,12(5):1020-1027