scholarly journals The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization.

1993 ◽  
Vol 4 (11) ◽  
pp. 1189-1204 ◽  
Author(s):  
M A Heine ◽  
M L Rankin ◽  
P J DiMario
Keyword(s):  
Nucleic Acid ◽  
Binding Activity ◽  
Full Length ◽  
Nucleic Acid Binding ◽  
Binding Assays ◽  
Nucleic Acid Probes ◽  
E Coli ◽  
Indirect Immunofluorescence Staining

Epitope-tagged Xenopus nucleolin was expressed in Escherichia coli cells and in Xenopus oocytes either as a full-length wild-type protein or as a truncation that lacked the distinctive carboxy glycine/arginine-rich (GAR) domain. Both full-length and truncated versions of nucleolin were tagged at their amino termini with five tandem human c-myc epitopes. Whether produced in E. coli or in Xenopus, epitope-tagged full-length nucleolin bound nucleic acid probes in in vitro filter binding assays. Conversely, the E. coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation maintained slight binding activity. Indirect immunofluorescence staining showed that myc-tagged full-length nucleolin properly localized to the dense fibrillar regions within the multiple nucleoli of Xenopus oocyte nuclei. The epitope-tagged GAR truncation also translocated to the oocyte nuclei, but it failed to efficiently localize to the nucleoli. Our results show that the carboxy GAR domain must be present for nucleolin to efficiently bind nucleic acids in vitro and to associate with nucleoli in vivo.

scholarly journals The MRG Domain Mediates the Functional Integration of MSL3 into the Dosage Compensation Complex

2005 ◽  
Vol 25 (14) ◽  
pp. 5947-5954 ◽  
Author(s):  
Violette Morales ◽  
Catherine Regnard ◽  
Annalisa Izzo ◽  
Irene Vetter ◽  
Peter B. Becker
Keyword(s):  
Nucleic Acid ◽  
X Chromosome ◽  
Dosage Compensation ◽  
De Novo ◽  
Functional Integration ◽  
Binding Activity ◽  
Nucleic Acid Binding ◽  
Dosage Compensation Complex

ABSTRACT The male-specific-lethal (MSL) proteins in Drosophila melanogaster serve to adjust gene expression levels in male flies containing a single X chromosome to equal those in females with a double dose of X-linked genes. Together with noncoding roX RNA, MSL proteins form the “dosage compensation complex” (DCC), which interacts selectively with the X chromosome to restrict the transcription-activating histone H4 acetyltransferase MOF (males-absent-on-the-first) to that chromosome. We showed previously that MSL3 is essential for the activation of MOF's nucleosomal histone acetyltransferase activity within an MSL1-MOF complex. By characterizing the MSL3 domain structure and its associated functions, we now found that the nucleic acid binding determinants reside in the N terminus of MSL3, well separable from the C-terminal MRG signatures that form an integrated domain required for MSL1 interaction. Interaction with MSL1 mediates the activation of MOF in vitro and the targeting of MSL3 to the X-chromosomal territory in vivo. An N-terminal truncation that lacks the chromo-related domain and all nucleic acid binding activity is able to trigger de novo assembly of the DCC and establishment of an acetylated X-chromosome territory.

Benzo(a)pyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo

Science ◽  
1976 ◽  
Vol 193 (4253) ◽  
pp. 592-595 ◽  
Author(s):  
I. Weinstein ◽  
A. Jeffrey ◽  
K. Jennette ◽  
S. Blobstein ◽  
R. Harvey ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Binding ◽  
Diol Epoxides

The mitotic phosphorylation of p54nrb modulates its RNA binding activity

2011 ◽  
Vol 89 (4) ◽  
pp. 423-433 ◽  
Author(s):  
Céline Bruelle ◽  
Mikaël Bédard ◽  
Stéphanie Blier ◽  
Martin Gauthier ◽  
Abdulmaged M. Traish ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Binding Assays ◽  
Non Coding Rna ◽  
Protein Partners ◽  
Biochemical Assays ◽  
Mitotic Phosphorylation

The RNA-binding protein p54nrb is involved in many nuclear processes including transcription, RNA processing, and retention of hyperedited RNAs. In interphase cells, p54nrb localizes to the nucleoplasm and concentrates with protein partners in the paraspeckles via an interaction with the non-coding RNA Neat1. During mitosis, p54nrb becomes multiphosphorylated and the effects of this modification are not known. In the present study, we show that p54nrb phosphorylation does not affect the interactions with its protein partners but rather diminishes its general RNA-binding ability. Biochemical assays indicate that in vitro phosphorylation of a GST-p54nrb construct by CDK1 abolishes the interaction with 5′ splice site RNA sequence. Site-directed mutagenesis shows that the threonine 15 residue, located N-terminal to the RRM tandem domains of p54nrb, is involved in this inhibition. In vivo analysis reveals that Neat1 ncRNA co-immunoprecipitates with p54nrb in either interphase or mitotic cells, suggesting that p54nrb–Neat1 interaction is not modulated by phosphorylation. Accordingly, in vitro phosphorylated GST-p54nrb still interacts with PIR-1 RNA, a G-rich Neat1 sequence known to interact with p54nrb. In vitro RNA binding assays show that CDK1-phosphorylation of a GST-p54nrb construct abolishes its interaction with homoribopolymers poly(A), poly(C), and poly(U) but not with poly(G). These data suggest that p54nrb interaction with RNA could be selectively modulated by phosphorylation during mitosis.

Nucleic-acid-binding properties of hnRNP-U/SAF-A, a nuclear-matrix protein which binds DNA and RNA in vivo and in vitro

1994 ◽  
Vol 221 (2) ◽  
pp. 749-757 ◽  
Author(s):  
Frank O. FACKELMAYER ◽  
Kirsten DAHM ◽  
Andrea RENZ ◽  
Uwe RAMSPERGER ◽  
Arndt RICHTER
Keyword(s):  
Nucleic Acid ◽  
Nuclear Matrix ◽  
Matrix Protein ◽  
Nucleic Acid Binding ◽  
Nuclear Matrix Protein ◽  
Binding Properties ◽  
Dna And Rna ◽  
Hnrnp U

scholarly journals Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Janine Weber ◽  
Han Bao ◽  
Christoph Hartlmüller ◽  
Zhiqin Wang ◽  
Almut Windhager ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleic Acid ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Point Mutations ◽  
Structural Basis ◽  
Binding Modes ◽  
Nucleic Acid Binding

The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

ChemInform Abstract: BENZO(A)PYRENE DIOL EPOXIDES AS INTERMEDIATES IN NUCLEIC ACID BINDING IN VITRO AND IN VIVO

1976 ◽  
Vol 7 (49) ◽  
pp. no-no
Author(s):  
I. B. WEINSTEIN ◽  
A. M. JEFFREY ◽  
K. W. JENNETTE ◽  
S. H. BLOBSTEIN ◽  
R. G. HARVEY ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Binding ◽  
Diol Epoxides

scholarly journals Amyloid aggregation of Streptococcus mutans Cnm influences its collagen-binding activity

2021 ◽  
Author(s):  
Nicholas de Mojana di Cologna ◽  
Sandip Samaddar ◽  
Carolina Valle ◽  
Jonathan A Vargas ◽  
Alejandro Aviles-Reyes ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Ex Vivo ◽  
In Silico Analysis ◽  
Binding Activity ◽  
Amyloid Aggregation ◽  
Binding Assays ◽  
Collagen Binding ◽  
Microtiter Plates

The glycosylated collagen- and laminin-binding surface adhesin Cnm is present in approximately 20% of S. mutans clinical isolates and is associated with systemic infections and increased caries risk. Other surface-associated collagen-binding proteins of S. mutans such as P1 and WapA have been demonstrated to form an amyloid quaternary structure with functional implications within biofilms. In silico analysis predicted that the b-sheet rich N-terminal collagen-binding domain (CBD) of Cnm has propensity for amyloid aggregation, whereas the threonine-rich C-terminal domain was predicted to be disorganized. In this study, thioflavin-T fluorescence and electron microscopy were used to show that Cnm forms amyloids either in its native glycosylated or recombinant non-glycosylated forms and that the CBD of Cnm is the main amyloidogenic unit of Cnm. We then performed a series of in vitro, ex vivo and in vivo assays to characterize the amylogenic properties of Cnm.  In addition, Congo red birefringence indicated that Cnm is a major amyloidogenic protein of S. mutans biofilms. Competitive binding assays using collagen-coated microtiter plates and dental roots, a substrate rich in collagen, revealed that Cnm monomers inhibit S. mutans binding to collagenous substrates whereas Cnm amyloid aggregates lose this property. Thus, while Cnm contributes to recognition and initial binding of S. mutans to collagen-rich surfaces, Cnm amyloid aggregation appears to represent a mechanism to modulate this activity in mature biofilms.

scholarly journals Origin Binding Activity of the Mycobacterial Plasmid pAL5000 Replication Protein RepB Is Stimulated through Interactions with Host Factors and Coupled Expression of repA

2002 ◽  
Vol 184 (8) ◽  
pp. 2204-2214 ◽  
Author(s):  
Abhijit Basu ◽  
Mamta Chawla-Sarkar ◽  
Santanu Chakrabarti ◽  
Sujoy K. Das Gupta
Keyword(s):  
Binding Activity ◽  
Complete Loss ◽  
Host Factors ◽  
Host Cells ◽  
High Concentration ◽  
E Coli ◽  
Specific Complex ◽  
Replication Region

ABSTRACT The minimal replication region of the mycobacterial plasmid pAL5000 encompasses the replication origin (ori) and two tandemly organized replication genes, repA and repB, the functions of which are not clearly known. It was observed that when the repA and repB genes were expressed in Escherichia coli, a strong ori binding activity was generated in the host cells. Inactivation of repB led to a complete loss of activity, whereas inactivation of repA had a partial effect, indicating that while repB plays an important role in the process, its activity is stimulated through coexpression of repA. However, this stimulatory effect could be demonstrated only when expression of repA and that of repB were coupled. At a relatively high concentration (1,000 nM), the purified RepB protein was found to form an ori complex with low specificity, which was sensitive to high salt concentrations and challenge by a nonspecific competitor. In contrast, the complex formed by an extract of repA-repB-expressing cells was highly specific and was resistant to both types of challenges. At a 10-fold-lower concentration, RepB did not exhibit ori binding activity, but it could nevertheless form a salt-resistant ori complex in vitro, provided that host factors were present. Antibody supershift experiments indicated that RepB is a key component of the specific complex formed by extracts prepared from E. coli cells expressing the repA and repB genes and also from mycobacterial cells harboring pAL5000-derived vectors. The results indicate that in vivo RepB interacts with host factors and forms an ori complex, but this activity is maximal only when there is coupled expression of repA.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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