A single-stranded nucleic acid-binding protein from Artemia salina. II. Interaction with nucleic acids.

Nowak, L; Marvil, D K; Thomas, John O.; Boublík, M.; Szer, Wlodzimierz

doi:10.1016/s0021-9258(18)43764-7

Cited by 28 publications

(4 citation statements)

References 22 publications

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“…hnRNP proteins can reassemble with hnRNA or synthetic polyribonucleotides in vitro, when subjected to dialysis against low salt buffer from an initial protein-RNA mixture prepared at high ionic strength (88). Similar results have been obtained with a crustacean protein that is homologous to vertebrate hnRNP core proteins (89). In vitro experiments provide useful information on the solution properties of these proteins (see also reference 7) but do not address the issue of whether or not hnRNP proteins bind specific hnRNA sequences in the cell.…”

Section: Hnrnp Proteins May Bind Specific Rna Sequencessupporting

confidence: 62%

Nuclear RNA-protein interactions and messenger RNA processing.

Pederson¹

1983

The Journal of Cell Biology

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Section: Hnrnp Proteins May Bind Specific Rna Sequencessupporting

confidence: 62%

Nuclear RNA-protein interactions and messenger RNA processing.

Pederson¹

1983

The Journal of Cell Biology

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“…This is indicative of disruption of the base stacking of the polymers by protein B23 (Karpel & Burchard, 1980). Lesser effects were seen with poly(U) (Figure 5C) as expected since this polymer exists predominantly as a random coil (Nowak et al, 1980). Filter binding assays indicated poly(U) was an effective competitor (data not shown), indicating that protein B23 binds to poly(U) and that the small effects were not due to the inability of B23 to bind the polymer.…”

Section: Resultsmentioning

confidence: 54%

“…For each sample, a minimum of nine spectra were accumulated and averaged by the computer. The binding mixtures contained 10 |iM (nucleotides) each polymer and varying concentrations of protein (0-10 Mg) in a binding buffer of 5 mM Tris-HCl, pH 7.4, 10 mM NaCl, and 10% glycerol (Nowak et al, 1980;Karpel & Burchard, 1980).…”

Section: Methodsmentioning

confidence: 99%

Interaction of nucleolar phosphoprotein B23 with nucleic acids

Dumbar¹,

Gentry²,

Olson³

1989

Biochemistry

153

111

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The interaction of eukaryotic nucleolar phosphoprotein B23 with nucleic acids was examined by gel retardation and filter binding assays, by fluorescence techniques, and by circular dichroism. All studies utilized protein prepared under native conditions by a newly developed purification procedure. Electrophoretic gel mobility shift assays with phage M13 DNA suggested that protein B23 is a single-stranded nucleic acid binding protein. This was confirmed in competition binding assays with native or heat-denatured linearized plasmid pUC18 DNA where the protein showed a marked preference for the denatured form. In other competition assays, there was no apparent preference for single-stranded synthetic ribo- versus deoxyribonucleotides. Equilibrium binding with poly(riboethenoadenylic acid) indicated cooperative ligand binding with a protein binding site size of 11 nucleotides and an apparent binding constant (K omega) of 5 x 10(7) M-1 which includes an intrinsic binding constant (K) of 6.3 x 10(4) M-1 and a cooperativity factor (omega) of 800. In circular dichroism (CD) studies, protein B23, when combined with the single-stranded synthetic nucleic acids poly(rA) and poly(rC), effected a decrease in ellipticity and a shift of the positive peak at 260-270 nm toward higher wavelengths, indicating helix destabilizing activity. No CD changes were seen with double-stranded poly(dA.dT). The change in ellipticity of poly(rA) was sigmoidal upon addition of protein, confirming the cooperative behavior seen with fluorescence methods. These studies indicate that protein B23 binds cooperatively with high affinity for single-stranded nucleic acids and exhibits RNA helix destabilizing activity. These features may be related to its role in ribosome assembly.

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“…At low protein to nucleic acid ratios, it will uniformly coat a nucleic acid molecule rather than form a globular subunit. At a high protein to nucleic acid ratio, HD40 will form polyparticles containing globular complexes of smaller diameter than 30S hnRNP and considerably more complexes appear to form on one ~X 174 molecule (26,34), indicating that the maximum number of nucleotides bound in one HD40 complex is much less than the 1,000 we have found for the vertebrate hnRNP particles.…”

Section: Discussionmentioning

confidence: 64%

Reconstitution of nucleoprotein complexes with mammalian heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins.

Pullman¹,

Martin²

1983

The Journal of Cell Biology

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Newly transcribed heterogeneous nuclear RNA (hnRNA) in the eucaryote cell nucleus is bound by proteins, giving rise to large ribonucleoprotein (RNP) fibrils with an inherent substructure consisting largely of relatively homogeneous -~20-nm 30S particles, which contain core polypeptides of 34,000-38,000 mol wt. To determine whether this group of proteins was sufficient for the assembly of the native beaded nucleoprotein structure, we dissociated 30S hnRNP purified from mouse ascites cells into their component proteins and RNA by treatment with the ionic detergent sodium deoxycholate and then reconstituted this complex by addition of Triton X-100 to sequester the deoxycholate. Dissociation and reassembly were assayed by sucrose gradient centrifugation, monitoring UV absorbance, protein composition, and radiolabeled nucleic acid, and by electron microscopy. Endogenous RNA was digested and reassembly of RNP complexes carried out with equivalent amounts of exogenous RNA or single-stranded DNA. These complexes are composed exclusively of groups of n 30S subunits, as determined by sucrose gradient and electron microscope analysis, where n is the length of the added nucleic acid divided by the length of nucleic acid bound by one native 30S complex (about 1,000 nucleotides). When the nucleic acid: protein stoichiometry in the reconstitution mixture was varied, only complexes composed of 30S subunits were formed; excess protein or nucleic acid remained unbound. These results strongly suggest that core proteins determine the basic structural properties of 30S subunits and hence of hnRNP. In vitro construction of RNP complexes using model nucleic acid molecules should prove useful to the further study of the processing of mRNA.Ultrastructural and biochemical lines of investigation have independently shown that heterogeneous nuclear RNA (hnRNA) is complexed with proteins throughout its existence in the cell nucleus (reviewed in reference 20). In electron micrographs of transcriptional complexes spread from lysed nuclei, hnRNA-protein complexes (hnRNP) frequently appear as fibrils consisting of 20-nm beads connected by RNA strands (1, 2, 21, 23). The biochemical entity isolated from purified nuclei by either isotonic extraction or mechanical disruption, referred to by us as "30S RNP" since it usually sediments more slowly than the 40S ribosomal subunit, is 18-25 nm in diameter and contains 700-1,000 nucleotides of rapidly labeled RNA and 40-60 copies of a group of four to six proteins of 34,000-40,000 mol wt with similar amino acid compositions and isoelectric points (pI = 8.0-9.0) (3,5,8, 11,17). These proteins are highly conserved throughout the vertebrates and turn over slowly in contrast with many other proteins (17-19). Since they only partially protect hnRNA from nuclease digestion (18), it has been suggested that these proteins form a core around which the nucleic acid is wrapped (3"0), and hence their designation as "core proteins." This hypothesis is attractive in that it allows for new transcripts to be mai...

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A single-stranded nucleic acid-binding protein from Artemia salina. II. Interaction with nucleic acids.

Cited by 28 publications

References 22 publications

Nuclear RNA-protein interactions and messenger RNA processing.

Nuclear RNA-protein interactions and messenger RNA processing.

Interaction of nucleolar phosphoprotein B23 with nucleic acids

Reconstitution of nucleoprotein complexes with mammalian heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins.

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