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We have investigated the structure of complexes formed between a series of poly(A)n (n = 30 to 480) and HD40 (helix-destabilizing protein, molecular weight of 40,000), the major protein component of 30 S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from the brine shrimp Artemia salina. Protein HD40 is similar to corresponding hnRNP proteins from higher eukaryotes and the complexes it forms with single-stranded nucleic acids are strikingly similar to the native "beads-on-a-string" structure of hnRNP. Using analytical ultracentrifugation and electron microscopy we find: (1) complexes formed between HD40 and long ribohomopolymers also have a beads-on-a-string structure, showing that the ability to form this structure is an inherent property of HD40, and is not dependent on any structural features of natural RNA; (2) complexes between HD40 and poly(A)160 form disks that are about 3 nm high by 18 nm in diameter and contain 20 HD40 molecules; (3) complexes of HD40 with poly(A)n with fewer than 160 nucleotides form sectors of a disk: 40 nucleotides give rise to a quarter of a disk, 80 nucleotides, half a disk, etc. The molecular weights increase with the size of poly(A)n at the rate of 5300 per nucleotide, a stoichiometry of eight nucleotides per HD40; (4) as the size of the poly(A)n increases beyond 160 nucleotides, the additional nucleoprotein elements may either initiate the formation of a second disk adjacent to the first or stack on top of the first disk to form a 6 nm high helix with a diameter of 18 nm. Based on these results, we propose that the existence of lateral protein-protein interactions that produce the basic 3 nm X 18 nm disk, combined with the marginal stability of the helix result in (a) interruptions of the helix that give rise to the beads-on-a-string appearance of the complexes, and (b) inherent heterogeneity of individual "beads" which may contain one or more turns of the helix. From measurements of HD40 complexes with coliphage MS2 RNA, phi X174 viral DNA as well as with the homopolymers, a bead is estimated to contain an average of approximately 300 nucleotides; approximately 1 X 8 turns of the helix.
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Binding of both synthetic poly(A) and naturally occurring poly(A) (+)mRNA as well as DNA to microtubule protein is mediated by microtubule-associated proteins; tubulin itself is not capable of binding these polymers. Bovine brain microtubule protein from immature animals was found to have a significantly lower capacity to bind poly(A) than microtubule protein from old animals. On the other hand, "old" microtubule protein binds DNA more efficiently than "immature" microtubule protein. Microtubule-associated protein 2 [preferred binding site for DNA] and tau proteins [preferred binding site for poly (A)] are specifically phosphorylated by a microtubule-associated, cAMP-dependent protein kinase. It was found that the affinity of microtubule protein for poly(A) is markedly decreased by autophosphorylation of the protein; in the case of DNA, the decrease in affinity was less. Autophosphorylation of "immature" microtubule proteins diminished the binding capacity for poly(A) to a greater extent than do "old" proteins. Scatchard plot analysis revealed that microtubule-protein possesses two different binding sites for poly(A). The corresponding dissociation constants were found to be increased in the phosphorylated system, but phosphorylation does not appear to alter the total number of binding sites. Compared to immature animals, microtubule protein from "old" bovine brains was found to have a reduced number of binding sites for poly(A), whereas the values of the dissociation constants remain unchanged. In contrast to total microtubule protein and homogeneous microtubule-associated protein 2, only one kind of binding site for poly(A) could be detected in homogeneous tau protein. No influence of different RNA or DNA species on microtubule protein-associated cAMP-dependent protein kinase, adenosine triphosphatase and guanosine triphosphatase activities could be detected.
Human trophoblast cultures were established from term placentae. Upon exposure to polyriboinosinic-polyribocytidylic acid (poly(I:C] the cultures produced interferon. Results of neutralization experiments and indirect immunofluorescence studies indicated that the trophoblast produces beta-interferon. The fusion of trophoblast cells into syncytia was accompanied by a restriction in interferon release after stimulation with poly(I:C). It was also demonstrated that the malignant choriocarcinoma cell line JAR produced less interferon than the non-transformed cytoorsyncytiotrophoblast.
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The interactions between basic oligopeptides (Lys2, Lys3, Arg2 and Arg3) and single stranded polynucleotides (poly(A), poly(C), poly(I) and poly(U) were investigated at low ion concentration by UV spectroscopy, circular dichroism and field jump relaxation. Various domains of binding were detected: 1) High concentrations (up to 1 mM) of some peptides induce opalescence followed by coacervation. Arg3 causes coacervation in all polynucleotides used, yet Lys3 only in poly(I). In the case of poly(I) the threshold concentration for coacervation is much lower for Arg3 (150 muM) than for Lys3 (500 muM). 2) Medium concentrations (greater than 10 muM) of Arg3 and Lys3 induce helix formation in poly(U). In the case of poly(I) cooperative helix formation is only induced by Lys3, but not by Arg3. 3) The onset of peptide association is observed at very low peptide concentrations (greater than or equal to 1muM) already by using the field jump method. The association is reflected by a relaxation process, that can be described by a single exponential within experimental accuracy. Measurements of relaxation time constants as a function of the peptide concentration provide information on the association constants K, the number of nucleotide residues per binding place n and the rate constants kR and kD. Using a simple model with independent and "separate" binding sites, K for Arg3 and Lys3 is found to be in the range of 10(6) to 10(7) M-1. In the case of Arg2 and Lys2 K is lower by a factor of about 10. For various polynucleotides KArg3 is only slightly higher than KLys3, except in the case of poly(I), where KArg3/KLys3 approximately 5. Similar data are obtained by application of a "sphere model" (see below). These results provide quantitative evidence for specific hydrogen bonding between the guanidino group of Arg and inosine. They also explain the absence of helix formation for poly(I) + Arg3: Arg blocks the hydrogen bonding sites of inosine. Thus cooperative coupling leads in this case to a considerable amplification of specificity in the peptide-polynucleotide interation. Both field jump and stopped flow data demonstrate a high mobility of the peptide ligands along the polymer, resulting in a redistribution being fast compared with the overall binding step. Based on this result the relaxation data are analysed by a "sphere" model, which considers a) excluded binding under the condition of fast ligand distribtuion along the lattice and b) the connection of sites into a polymer sphere. The rate constants obtained by this model are in the range of 4 X 10(11) M-1 s-1. These high values reflect the large reaction distance for polymers of chain lengths around 1000. A comparison with rate constants obtained previously for oligomer complexes indicates that the recombination rate is approximately a function of the square root of the nucleotide chain length, which is directly related to the mean radius of coiled polymers.
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