scholarly journals Viroplasm Protein P9-1 ofRice Black-Streaked Dwarf VirusPreferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

2013 ◽  
Vol 87 (23) ◽  
pp. 12885-12899 ◽  
Author(s):  
Jianyan Wu ◽  
Jia Li ◽  
Xiang Mao ◽  
Weiwu Wang ◽  
Zhaobang Cheng ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Rna Binding ◽  
Structure And Function ◽  
Interior Structure ◽  
Mobility Shift ◽  
Chromatography Analysis ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
And Function

The P9-1 protein ofRice black-streaked dwarf virus(RBSDV) is an essential part of the viroplasm. However, little is known about its nature or biological function in the viroplasm. In this study, the structure and function of P9-1 were analyzed forin vitrobinding to nucleic acids. We found that the P9-1 protein preferentially bound to single-stranded versus double-stranded nucleic acids; however, the protein displayed no preference for RBSDV versus non-RBSDV single-stranded ssRNA (ssRNA). A gel mobility shift assay revealed that the RNA gradually shifted as increasing amounts of P9-1 were added, suggesting that multiple subunits of P9-1 bind to ssRNA. By using discontinuous blue native gel and chromatography analysis, we found that the P9-1 protein was capable of forming dimers, tetramers, and octamers. Strikingly, we demonstrated that P9-1 preferentially bound to ssRNA in the octamer, rather than the dimer, form. Deletion of the C-terminal arm resulted in P9-1 no longer forming octamers; consequently, the deletion mutant protein bound to ssRNA with significantly lower affinity and with fewer copies bound per ssRNA. Alanine substitution analysis revealed that electropositive amino acids among residues 25 to 44 are important for RNA binding and map to the central interior structure that was formed only by P9-1 octamers. Collectively, our findings provide novel insights into the structure and function of RBSDV viroplasm protein P9-1 binding to RNA.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

scholarly journals Characterization of the RNA-Binding Domains in the Replicase Proteins of Tomato Bushy Stunt Virus

2003 ◽  
Vol 77 (17) ◽  
pp. 9244-9258 ◽  
Author(s):  
K. S. Rajendran ◽  
Peter D. Nagy
Keyword(s):  
Rna Binding ◽  
Tomato Bushy Stunt Virus ◽  
Binding Motif ◽  
Mobility Shift ◽  
Terminal Domain ◽  
Binding Domains ◽  
Replicase Proteins ◽  
Rna Binding Domains ◽  
Gel Mobility Shift

ABSTRACT Tomato bushy stunt virus (TBSV), a tombusvirus with a nonsegmented, plus-stranded RNA genome, codes for two essential replicase proteins. The sequence of one of the replicase proteins, namely p33, overlaps with the N-terminal domain of p92, which contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its nonoverlapping C-terminal portion. In this work, we demonstrate that both replicase proteins bind to RNA in vitro based on gel mobility shift and surface plasmon resonance measurements. We also show evidence that the binding of p33 to single-stranded RNA (ssRNA) is stronger than binding to double-stranded RNA (dsRNA), ssDNA, or dsDNA in vitro. Competition experiments with ssRNA revealed that p33 binds to a TBSV-derived sequence with higher affinity than to other nonviral ssRNA sequences. Additional studies revealed that p33 could bind to RNA in a cooperative manner. Using deletion derivatives of the Escherichia coli-expressed recombinant proteins in gel mobility shift and Northwestern assays, we demonstrate that p33 and the overlapping domain of p92, based on its sequence identity with p33, contain an arginine- and proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). This motif is highly conserved among tombusviruses and related carmoviruses, and it is similar to the arginine-rich motif present in the Tat trans-activator protein of human immunodeficiency virus type 1. We also find that the nonoverlapping C-terminal domain of p92 contains additional RNA-binding regions. Interestingly, the location of one of the RNA-binding domains in p92 is similar to the RNA-binding domain of the NS5B RdRp protein of hepatitis C virus.

scholarly journals Bacillus subtilis Aconitase Is Required for Efficient Late-Sporulation Gene Expression

2006 ◽  
Vol 188 (17) ◽  
pp. 6396-6405 ◽  
Author(s):  
Alisa W. Serio ◽  
Kieran B. Pechter ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Activation ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Binding Activity ◽  
High Catalytic Activity ◽  
Spore Coat ◽  
Mobility Shift ◽  
Gel Mobility Shift

ABSTRACT Bacillus subtilis aconitase, encoded by the citB gene, is homologous to the bifunctional eukaryotic protein IRP-1 (iron regulatory protein 1). Like IRP-1, B. subtilis aconitase is both an enzyme and an RNA binding protein. In an attempt to separate the two activities of aconitase, the C-terminal region of the B. subtilis citB gene product was mutagenized. The resulting strain had high catalytic activity but was defective in sporulation. The defect was at a late stage of sporulation, specifically affecting expression of σK-dependent genes, many of which are important for spore coat assembly and require transcriptional activation by GerE. Accumulation of gerE mRNA and GerE protein was delayed in the aconitase mutant strain. Pure B. subtilis aconitase bound to the 3′ untranslated region of gerE mRNA in in vitro gel mobility shift assays, strongly suggesting that aconitase RNA binding activity may stabilize gerE mRNA in order to allow efficient GerE synthesis and proper timing of spore coat assembly.

scholarly journals MSY2 and MSY4 Bind a Conserved Sequence in the 3′ Untranslated Region of Protamine 1 mRNA In Vitro and In Vivo

2001 ◽  
Vol 21 (20) ◽  
pp. 7010-7019 ◽  
Author(s):  
Flaviano Giorgini ◽  
Holly G. Davies ◽  
Robert E. Braun
Keyword(s):  
Untranslated Region ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Binding Activity ◽  
Mobility Shift ◽  
Conserved Sequence ◽  
Mobility Shift Assay ◽  
Protamine 1

ABSTRACT Y-box proteins are major constituents of ribonucleoprotein particles (RNPs) which contain translationally silent mRNAs in gametic cells. We have recently shown that a sequence-specific RNA binding activity present in spermatogenic cells contains the two Y-box proteins MSY2 and MSY4. We show here that MSY2 and MSY4 bind a sequence, 5′-UCCAUCA-3′, present in the 3′ untranslated region of the translationally repressed protamine 1 (Prm1) mRNA. Using pre- and post-RNase T1-digested substrate RNAs, it was determined that MSY2 and MSY4 can bind an RNA of eight nucleotides containing the MSY2 and MSY4 binding site. Single nucleotide mutations in the sequence eliminated the binding of MSY2 and MSY4 in an electrophoretic mobility shift assay, and the resulting mutants failed to compete for binding in a competition assay. A consensus site of UACCACAUCCACU(subscripts indicate nucleotides which do not disrupt YRS binding by MSY2 and MSY4), denoted the Y-box recognition site (YRS), was defined from this mutational analysis. These mutations in the YRS were further characterized in vivo using a novel application of the yeast three-hybrid system. Experiments with transgenic mice show that disruption of the YRS in vivo relieves Prm1-like repression of a reporter gene. The conservation of the RNA binding motifs among Y-box protein family members raises the possibility that other Y-box proteins may have previously unrecognized sequence-specific RNA binding activities.

scholarly journals Diastereomeric Recognition of 5',8-cyclo-2'-deoxyadenosine Lesions by Human poly(ADP-ribose) Polymerase 1 in Biomimetic Model

2018 ◽  
Author(s):  
Annalisa Masi ◽  
Arianna Sabbia ◽  
Carla Ferreri ◽  
Francesco Manoli ◽  
Yanhao Lai ◽  
...  
Keyword(s):  
Excision Repair ◽  
Strand Break ◽  
Dna Lesions ◽  
Affinity Constant ◽  
Mobility Shift ◽  
New Finding ◽  
Mobility Shift Assay ◽  
Low Efficiency ◽  
Gel Mobility Shift

Abstract5′,8-Cyclo-2′-deoxyadenosine (cdA), in the 5′R and 5′Sdiastereomeric forms, are typical non strand-break oxidative DNA lesions, induced by hydroxyl radicals, with emerging importance as a molecular marker. These lesions are exclusively repaired by nucleotide excision repair (NER) mechanism with a low efficiency, thus readily accumulating in the genome. Poly(ADP-ribose) polymerase1 (PARP1) acts as an early responder to DNA damage and plays a key role as a nick sensor in the maintenance of the integrity of the genome by recognizing nicked DNA. So far, it was unknown whether the diastereomeric cdA lesions could induce specific PARP1 binding. Here we provide the first evidence of PARP1 to selectively recognize the diastereomeric lesions 5′S-cdA and 5′R-cdA in vitro as compared to deoxyadenosine in model DNA substrates (23-mers) by using circular dichroism,fluorescence spectroscopy, immunoblotting analysis and gel mobility shift assay. Several features of the recognition of the damaged and undamaged oligonucleotides by PARP1were characterized. Remarkably, PARP1 efficiently binds to both cdA lesions in the double stranded (ds)-oligonucleotides. In particular, PARP1 proved to bind 5′S-cdAwith a higher affinity constant for the 5'S lesion in a model of ds DNA than 5′R-cdA, showing different recognition patterns, also compared with undamaged dA. This new finding highlights the ability of PARP1 to recognize and differentiate the distorted DNA backbone in a biomimetic system caused by different diastereomeric forms of a cdA lesion.

scholarly journals Reovirus Protein ςNS Binds in Multiple Copies to Single-Stranded RNA and Shares Properties with Single-Stranded DNA Binding Proteins

2000 ◽  
Vol 74 (13) ◽  
pp. 5939-5948 ◽  
Author(s):  
Anne Lynn Gillian ◽  
Stephen C. Schmechel ◽  
Jonathan Livny ◽  
Leslie A. Schiff ◽  
Max L. Nibert
Keyword(s):  
Dna Binding ◽  
Nucleic Acids ◽  
Binding Proteins ◽  
Atp Hydrolysis ◽  
Nonstructural Protein ◽  
Dna Binding Proteins ◽  
Mobility Shift ◽  
Single Stranded Dna ◽  
Gel Mobility Shift

ABSTRACT Reovirus nonstructural protein ςNS interacts with reovirus plus-strand RNAs in infected cells, but little is known about the nature of those interactions or their roles in viral replication. In this study, a recombinant form of ςNS was analyzed for in vitro binding to nucleic acids using gel mobility shift assays. Multiple units of ςNS bound to single-stranded RNA molecules with positive cooperativity and with each unit covering about 25 nucleotides at saturation. The ςNS protein did not bind preferentially to reovirus RNA over nonreovirus RNA in competition experiments but did bind preferentially to single-stranded over double-stranded nucleic acids and with a slight preference for RNA over DNA. In addition, ςNS bound to single-stranded RNA to which a 19-base DNA oligonucleotide was hybridized at either end or near the middle. When present in saturative amounts, ςNS displaced this oligonucleotide from the partial duplex. The strand displacement activity did not require ATP hydrolysis and was inhibited by MgCl2, distinguishing it from a classical ATP-dependent helicase. These properties of ςNS are similar to those of single-stranded DNA binding proteins that are known to participate in genomic DNA replication, suggesting a related role for ςNS in replication of the reovirus RNA genome.

scholarly journals Asymmetric dimerization of adenosine deaminase acting on RNA facilitates substrate recognition

2020 ◽  
Vol 48 (14) ◽  
pp. 7958-7972 ◽  
Author(s):  
Alexander S Thuy-Boun ◽  
Justin M Thomas ◽  
Herra L Grajo ◽  
Cody M Palumbo ◽  
SeHee Park ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Structure ◽  
Rna Binding ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Sequence Elements ◽  
Dimerization Interface ◽  
Gel Mobility Shift ◽  
And Function

Abstract Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.

Human Myeloma Cells Express the Bone Regulating Gene Runx2/Cbfa1 and Produce Osteopontin That It Is Involved in Myeloma-Induced Angiogenesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2344-2344
Author(s):  
Nicola Giuliani ◽  
Simona Colla ◽  
Francesca Morandi ◽  
Sabrina Bonomini ◽  
Monica Crugnola ◽  
...  
Keyword(s):  
Bone Matrix ◽  
Myeloma Cell ◽  
Mobility Shift ◽  
Myeloma Cells ◽  
Vessel Formation ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
Rpmi 8226 ◽  
Human Myeloma Cells

Abstract Osteopontin (OPN) is a multifunctional bone matrix glycoprotein that it is involved in angiogenesis, cell survival and tumor progression. OPN gene expression by human cells is mainly regulated by the bone specific trascription factor Runx2 namely also CBFA1 (Runx2/Cbfa1) even if other transcription factors may be also involved in the production of OPN by cancer cells as the CCAAT/enhancer-binding protein alpha (C/EBPα) and AML-1. In this study we show that human myeloma cell lines (HMCLs): RPMI-8226, U266, XG-1 and XG-6 but not ARH-77 and normal CD19+ cells directly produce OPN and express its major regulating gene Runx2/Cbfa1. The activity of Runx2/Cbfa1 in human myeloma cells has been also demonstrated by a gel mobility shift assay. On the other hand we found that all HMCLs tested were negative for C/EBPα mRNA, whereas AML-1A and AML-1B mRNA were expressed in all HMCLs even if any correlation has not been found between OPN expression and AML-1A/AML-1B ratio. In addition we found that OPN production by myeloma was up-regulated at both mRNA and protein level by IL-6 and IGF-I through a Runx2/Cbfa1 mediated mechanism; in turn recombinant human OPN was able to increase myeloma cells proliferation. The potential role of OPN in MM-induced angiogenesis has been also investigated in an experimental model of angiogenesis. rhOPN treatment stimulated vessel formation as compared to control and the conditioned medium (CM) of HMCLs, significantly increased vessel formation in comparison either with control or with VEGF treatment. On the contrary OPN-immunodepleted CM of HMCLs had not a stimulatory effect on vessel formation and the presence of anti OPN Ab inhibited vessel formation induced by HMCLs. The expression of OPN by purified bone marrow (BM) CD138+ cells has been also investigated in 60 newly diagnosed multiple myeloma (MM) patients, finding that 40% of MM patients tested expressed OPN. Higher OPN levels have been detected in the BM plasma of MM patients positive for OPN as compared to control subjects. A significant higher MVD was observed in the group of patients positive for OPN, (mean±SE: 29.1±0.7 vs. 17.55±0.37; p<0.01) and similarly, the number of microvessels per field was higher in OPN positive patients in comparison with OPN negative ones (mean±SE: 6.7±0.15 vs. 4.28±0.04; p=0.05). In conclusion our data highlight the direct ectopic production of OPN by human myeloma cells with a Runx2/CBFA1 mediated mechanism and the capacity of myeloma-derived OPN to stimulate angiogenesis in vitro. In addition OPN has been detected in a subset of MM patients with higher BM angiogenesis suggesting its potential involvement in pathophysiology of MM-induced angiogenesis.

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