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An analysis of the published data of the electrophoresis of the products of silk fibroin exhibiting discontinuities or pauses in the translational process showed that about 60 times of pauses occur during the translation of the mRNA from silk fibroin. In addition, we estimated that silk fibroin gene as a whole (15kb) is composed of about 60 alternate, crystalline-noncrystalline, arrays of nucleotide sequence elements each connected by the boundary sequence, on the basis of the nucleotide sequence of the partial cDNA clone of the heavy chain gene of Bombyx mori available in the literature. The coincidence of the total number of pauses with that of the alternate arrays suggests that one translational pausing site exists in each of the unit (alternate array + boundary ) nucleotide sequence in the silk fibroin mRNA template.

Discontinuous translational elongation of polypeptides is observed during spider dragline silk fibroin synthesis (1,2). The repeating segment of one of the two subunit proteins constituting spider major ampullate (dragline) silk of Nephila clavipes, Spidroin 2, consists of alternate alanine-rich and proline-rich regions (3). It was found that the calculated free energy of the secondary structure of Spidroin 2 mRNA per nucleotide for the alanine-rich region is about the same as that for the successive proline-rich region. The small stability changes of local mRNA secondary structures along the mRNA chain suggest that the translational pauses observed for dragline silk fibroin synthesis may not be correlated with Spidroin 2 mRNA structure, in contrast to Spidroin 1 mRNA structure which may explain the translational pauses (4).

A stability map of local secondary structure of the mRNA of the triple-helical alpha 2(I) chain domain of chicken type I collagen was obtained by plotting the free energy of the optimal secondary structure of a local segment in mRNA against the segment position along a base sequence of the mRNA. It was found that the positions of the minima of free energy in the plot coincide with the positions where synthesis pauses of the alpha-chain polypeptides of the corresponding sizes translated from the mRNA have been reported to occur (1). The codon usage pattern of each of the three major amino acids of the alpha-chain domain of the collagen, Gly, Pro and Ala, fluctuates considerably along the base sequence segments of the mRNA and a deviation of the pattern from that of the average of the whole alpha 2(I) chain domain mRNA, particularly for Gly codons, leads to a loss of the stability of the local secondary structure of the mRNA. The results suggest that selection has operated on the codon usage to optimize the secondary structure characteristic of the mRNA of the chicken collagen alpha 2(I) chain domain which leads to a nonuniform polypeptide elongation pattern.

The 13C nuclear magnetic resonance studies have been carried out on histones H1 and H5, by focusing our interest on possible formation of specific salt bridges between acidic and basic amino acid residues in the proteins and also on the structural difference between the two proteins. The 13C chemical shift and pKa values of the carboxyl group of glutamic acid residues in the histones coincided with those of free glutamic acid. Based on this result and another experiment using completely modified lysine residues in the histones, no evidence for a specific interaction between acidic and basic residues has been found. It has also been shown that the pH-effects of aliphatic and aromatic resonances are quite different between H1 and H5, suggesting that the globular domain of H5 is more stable than that of H1. The correlation time (1.5 ns) for the alpha-carbons of H5 estimated from 13C nuclear Overhauser enhancement was twice as long as that of H1 (0.9 ns), indicating that the backbone in the N-terminal and C-terminal domains of H5 is less mobile than that of H1.

The structure of the nucleosome core particle at low ionic strenth (10-0.04 mM Na + ) has been studied by small angle neutron scattering in various H 2 O/D 2 O mixtures. At 0.04 mM Na + ,the radius of gyration obtained at 39% D 2 O where scattering from the DNA dominates was 32 A, and at 65% D 2 O where scattering from the histones dominates, 40 A. An abrupt increase in the radius of gyration from 35 to 40 A was observed at about 1 mM ionic strength at 65% D 2 O, with decreasing the ionic strength from 10 mM. No loss of the histone secondary structure was detected by circular dichroism over the range of the ionic strength examined. These results suggest that at low ionic strength (⪅1 mM) the histones are located outside of the nucleosome core particle accompanied by an alteration of the tertiary and/or quaternary structure of the histone octamer, with the DNA in a folded conformation.

The arginine and lysine residues of calf thymus histone H1 were modified with large molar excesses of 2,3-butanedione and O-methylisourea, respectively. Kinetic study of the modification reaction of the arginine residue revealed that the reaction is divided into the two pseudo-first-order processes. About a third (1 Arg) of the total arginine residues of the H1 molecule was rapidly modified without causing any detectable structural change of the molecule, and the slow modification of the remaining arginine residues (2 Arg) led to a loss of the folded structure of H1. In the case of lysine residue modification, 93% (56 Lys) of the total lysine residues of the H1 was modified with the same rate constant, while 7% (4 Lys) of lysine residue remained unmodified. When the reaction was performed in the presence of 6M guanidine-HCl, all of lysine residues were modified. It is concluded that the 2 arginine and 4 lysine residues resistant to modification are buried in interior regions of the H1 molecule and play an important role in the formation of the H1 globular structure, while the other 1 arginine and 56 lysine residues are exposed to solvent.

We have identified two distinctive regions of the repetitive unit nucleotide sequence of fibroin mRNA of Bombyx mori. The codon usage for the major amino acids, glycine, alanine and serine is distinctly different in these two regions, indicating that it is determined by the fibroin mRNA or gene structure but not by the tRNA population. Comparative computer analyses of nucleotide substitutions in the unit sequence suggest that selection has operated on the codon usage to optimize the secondary structure characteristic of the fibroin mRNA.

Distinct differences were found between DNA—polylysine and DNA—polyarginine complexes formed under identical conditions of salt-gradient dialysis in the change of the CD spectrum as a function of salt concentrations. Based on the findings, the CD and the induced optical activities of the bound dye of the DNA—polylysine—polyarginine complex, formed by the step-gradient dialysis procedure from 5 M urea—2 M NaCl, were investigated in order to evaluate the relative contributions of polylysine and polyarginine to the structural changes of DNA. It was shown that the two polypeptides do not act additively in changing the CD spectrum of DNA in the DNA—polylysine—polyarginine complex, but a small amount of polyarginine is effectively able to block large spectral changes induced by polylysine throughout the NaCl concentration range examined (1–0.01 M). It appears that the most likely effect of polyarginine binding on DNA—polylysine complex is to break down a regularity of the aggregated structure characterized by its large and anomalous CD. Acridine orange, bound to the peptide-free portions in the DNA—polylysine—polyarginine complex, exhibited characteristic extrinsic Cotton effects differing from either of the DNA—polylysine—dye or the DNA—polyarginine—dye system.

Circular dichroism spectroscopy has been used to investigate the influence of polylysine and polyarginine on the transition to a condensed state of DNA brought about by high concentrations of polyethyleneglycol and salt. From the dependence on DNA concentration of the CD signals, the anomalous CD of free DNA in polyethyleneglycol salt solution was attributed to the intermolecular association of DNA molecules. The CD spectral changes in polyethyleneglycol salt solution of the DNA · polylysine complex were indistinguishable from those of free DNA while the DNA · polyarginine complex suffered much smaller spectral changes as compared with free DNA, at low DNA concentrations where time-independent CD spectra were observed in polyethyleneglycol salt solution for both the complexed and free DNA. The repression of the spectral change by the latter complex was more remarkable at higher ratios of polyarginine to DNA. The facts indicate that, whereas polylysine binding has little influence on the intermolecular structural transition of double-stranded DNA into a compact molecular configuration in polyethyleneglycol salt solution, polyarginine binding has an effect of inhibiting the transition.

The interaction between DNA and ionen polymers, -[N+(CH3)2(CH2)mN+(CH3)2(CH2)n], with m-n of 3–3, 6–6, and 6–10 were examined in order to know how the binding behavior of cationic polymers with DNA depends on the charge density of polycation. The ionen polymer has no bulky side chain and the binding forces with DNA would be attributed mainly to electrostatic interaction. When 3–3 ionen polymers were added to DNA solution, precipitable complexes with the ratio of cationic residue to DNA phosphate (+/−) of 1/1 and the free DNA molecules were segregated, while 6–6 and 6–10 ionen polymers formed soluble complexes with DNA molecules up to (+/−) = 0.5. This suggests that 3–3 ionen polymers bind cooperatively with DNA while 6–6 and 6–10 ionen polymers bind noncooperatively. The cooperative binding of 3–3 ionen polymer and the noncooperative binding of 6–6 ionen polymer were also supported by the thermal melting and recooling profiles from the midpoint between first and second meltings. It was concluded that the charge density of DNA phosphate is a critical value determining whether the ionen polymers bind to DNA by a cooperative or by a noncooperative binding, since the distance between successive cationic charges of 3–3 ionen polymer is shorter than that between successive phosphate charges on DNA double helix and those of 6–6 and 6–10 ionen polymers are longer.

The posterior silk glands of Bombyx mori are highly differentiated to produce fibroin, the specific cocoon protein. We have now succeeded in isolating the nuclei from the posterior silk gland cells, and a giant arborescent nucleus with a strikingly complicated morphology was observed. Biochemical analysis of the composition of the nuclei confirmed the existence of the four core histones in equimolar quantities and H1 histone. The isolated nuclei obtained here might be prerequisite for the study of the structure of the giant polyploid nucleus and its relation to the tissue-specific fibroin gene expression.

The conformational transition of poly(L-agrignine) by binding with various mono-, di-, and polyvalent anions, especially with SO, was studied by CD measurements. The intramolecular random coil-to-α-helix conformational transition and the subsequent transition to the β-turn-like structure was caused by binding with SO. The binding data obtained from equilibrium dialysis experiments showed that the α-helical conformation of poly(L-arginine) is stabilized at a 1:3 stoichiometric ratio of bound SO to arginine residue; at higher free SO concentrations, the α-helix converts to the β-turn-like structure accompanied by a decrease in amount of bound SO. The same conformaitonal transition of poly(L-arginine) also occurred in the solutions of other divalent anions (SO, CO, and HPO) and polyvalent anions (P2O, P3O). Among the monovalent anions examined, CIO and dodecyl sulfate were effective in including α-helical conformation, while the other monovalent anions (OH−, Cl−, F−, H2PO, HCO and CIO) failed to induce poly(L-arginine) to assume the α-helical conformation. Thus, we noticed that, except for dodecyl sufate, the terahedral structure is common to the α-helix-forming anions. A well-defined model to the α-helical poly(L-arginine)/anion complex was proposed, in which both the binding stoichiometry of anions to the arginine residue and the tetrahedral structure of anions were taken into consideration. Based on these results, it was concluded that the tetrahedral-type anions stabilize the α-helical conformation of poly(L-arginine) by crosslinking between two guanidinium groups of nearby side chains on the same α-helix through the ringed structures stabilized by hydrogen bonds as well as by electrostatic interaction. Throughout the study it was noticed that the structural behavior of poly(L-arginine) toward anions is distinct from that of poly(L-lysine).

Poly(L-arginine) assumes the α-helix in the presence of the tetrahedral-type anions or some polyanions by forming the “ringed-structure bridge” between guanidinium groups and anions which is stabilized by a pair of hydrogen bonds and electrostatic interaction [Ichimura, S., Mita, K. & Zama, M. (1978) Biopolymers17, 2769–2782; Mita, K., Ichimura, S. & Zama, M. (1978) Biopolymers17, 2783–2798]. This paper describes the parallel CD studies on the conformational effects on poly (L-homoarginine) of various mono-, di-, polyvalent anions and some polyanions, as well as alcohol and sodium dodecylsulfate. The random coil to α-helix transition of poly(L-homoarginine) occurred only in NaClO4 solution or in the presence of high content of ethanol or methanol. The divalent and polyvalent anions of the tetrahedral type (SO, HPO, and P2O), which are strong α-helix-forming agents for poly(L-arginine), failed to induce the α-helical conformation of poly(L-homoarginine). By complexing with poly(L-glutamic acid) or with polyacrylate, which is also a strong α-helix-forming agent for poly(L-arginine), poly(L-homoarginine) only partially formed the α-helical conformation. Monovalent anions (OH−, Cl−, F−, and H2PO) did not change poly(L-homoarginine) to the α-helix, and in the range of pH 2–11, the polypeptide remained in an unordered conformation. In sodium dodecylsulfate, poly(L-homoarginine) exhibited the remarkably enlarged CD spectrum of an extended conformation, while poly(L-arginine) forms the α-helix by interacting with the agent. Thus poly(L-homoarginine), compared with poly(L-arginine), has a much lower ability to form the α-helical conformation by interacting with anions. The stronger hydrophobicity of homoarginine residue in comparison with the arginine residue would provide unfavorable conditions to maintain the α-helical conformation.

The induced circular dichroism (CD) in the visible region of acridine orange bound to the double-stranded RNA from cytoplasmic polyhedrosis virus and to yeast tRNA has been measured as a function of RNA phosphate-to-dye ratio (P/D), under the conditions of 0.01 M Na+ at pH 7.0. The shape of the CD spectrum of acridine orange bound to the double-stranded RNA was quite different from the spectrum of the dye bound to DNA. The CD spectral features of acridine orange bound to the double-stranded regions in tRNA closely resembled those of the double-stranded RNA-dye complex, suggesting that the dyes bind similarly to the two RNA's. It was further found that the CD spectrum of the tRNA-dye complex at sufficiently high P/D ratios, which is assignable to monomeric, intercalated dye to the base-paired parts in tRNA, is also distinct from the corresponding spectrum of the DNA-dye complex.

The structural behavior of the nucleosome core particles in the range of solvent Na+ concentration from 10.45 to 0.45 mM has been studied by small-angle neutron and synchroton radiation X-ray scattering, sedimentation, atomic absorption spectroscopy, density measurements, and circular dichroism. With decreasing salt concentration, the appearance of a scattering peak that is assignable to interparticle interactions, an intraparticle structural transition, a decrease in the sedimentation velocity of the particle, and a release of bound Na+ ions from the particle are all observed concurrently when the ratio of solvent Na+ ions per particle is below approximately 1000. These observations are interpreted to indicate that a release of bound Na+ ions from the particle brings about structural rearrangements and weakens the electrostatic shielding of the particle, and this introduces long-range repulsive ordering of the particle in low-salt solution. Analyses of the scattering data indicate that the rearrangement within the core particle in low-salt solution is slight, changing the particle's shape slightly from cylindrical to a more spherical form by moving the center of the mass of the DNA somewhat inward with accompanying small decreases in the radii of gyration of both the DNA and the histones.

Comparative studies on the conformational stability of histones H1 and H5 have been carried out by monitoring the pH-induced conformational transitions of the proteins by CD and 1H NMR spectroscopies. The transition point of H1 agrees with the pKa of the carboxyl groups of the acidic residues. In contrast, the transition of H5 is associated with the ionization of the histidine residues which exist exclusively in H5, as well as the deionization of the acidic residues. These observations, combined with the result of the deuterium exchange rates of the histidine C-2 protons, led us to conclude that His-25 and His-62, which are buried in the globular domain, play an important role in the conformational stability of histone H5.

A method for the quantitative determination of single-stranded sections in DNA was developed. Fractions of denatured sections can be estimated using a suitable calibration curve which is obtained on the basis that acridine orange bound to denatured DNA emits a weaker fluorescence at 530 nm and a stronger one at 640 nm than does the dye bound to native DNA. The most suitable conditions, where dye is bound to both types of DNA with the same efficiency, were determined to be an ionic strength of 0.01 and a DNA phosphate to bound dye ratio of 7.5. Under these conditions, the fluorescence intensity at 530 nm of the dye added to the mixture of native and heat-denatured DNA decreased linearly as the fraction of denatured DNA increased. In relation to making a calibration curve, the conformation of heat-denatured calf thymus DNA at an ionic strength of 0.01 was studied. When heated DNA was chilled, only a few percent of total nucleotides reformed the double helical structure in spite of the considerable decrease in hyperchromicity.

The strong binding of acridine orange with DNA was studied by means of equilibrium dialysis and thermal renaturation of the complex. Thermodynamic parameters for the strong binding with native and denatured DNA were calculated from the data of equilibrium dialysis. The association constant and the change of free energy were nearly equal for both types of DNA, but the changes of enthalpy and entropy were quite different. Binding to both types of DNA may be strong binding in nature, but the accompanying effects on the secondary structures of DNA would be different. It was found that the presence of acridine orange inhibits the completion of renaturation, presumably due to a stabilizing effect of the bound dye on the “wrong” base pairs as they are formed. The results support the notion that binding of acridine orange with the double helix is an intercalation into adjacent base pairs, while the binding with single strand is a partial intercalation into adjacent bases.

Soluble complexes of DNA with poly-L-lysine and poly-L-arginine were prepared by dialysis or mixing method, and their circular dichroism spectra and viscosities were measured. Both complexes formed by dialysis method had different circular dichroism spectra from that of free DNA respectively, i.e., the spectrum of the DNA-poly-L-lysine complex was similar to that of DNA at high salt concentration and the spectrum of the DNA-poly-L-arginine complex resembled that of DNA under melting at elevated temperature. The viscosity of the both complexes decreased pronouncedly with the increase in peptide to DNA ratio, although the viscosity of the DNA-poly-L-lysine complex was more rapidly decreased than that of the DNA-poly-L-arginine complex. The difference between the two polypeptides in changing DNA structure was observed also in the complexes formed by mixing method.

The circular dichroism (CD) spectra of DNA–acridine orange (DNA–AO) complex in the visible region were measured at DNA phosphate-to-dye ratios (P/D) from 1 to 550. The CD spectrum of DNA–AO complex in the P/D ratio between 1 and approximately 40 consists of four components, i.e., positive CD bands centered at 510 and 480 mμ, and negative CD bands at 497 and 468 mμ. The CD bands at 510 and 468 mμ are optimum at P/D = 4, and the change of e1 − er with P/D suggests that both of them are induced from the interaction between dye molecules bound to adjacent DNA binding sites, each of which is composed of four nucleotides. This is supported by the fact that the values of e1 − er for both decrease with increasing temperature or increasing methylene blue concentration added to the complex. The negative Cotton effect at, 497 mμ is most favored at larger P/D ratio (∼8), and the suggested assignment is to the interaction between two dye molecules bound with an empty site between them. A positive Cotton effect at 480 mμ is observed at P/D ratio of less than 4 and is optimum at 1. Above P/D ratio of 40, the CD spectrum of the complex can not be resolved into its components and even at sufficiently high P/D ratio (550) the complex shows a small Cotton effect.

Nucleosome cores mixed with the high mobility group proteins, HMG1 and HMG2, in 2 M NaCl, 5 M urea, 0.2 mM EDTA and 10 mM Tris pH 7.0, have been reconstituted by salt gradient dialysis. The reconstituted material, in 10 mM Tris pH 7.0, had a sedimentation peak at the same position as that of control nucleosome cores in sucrose density gradient ultracentrifugation. The SDS polyacrylamide gel electrophoresis of the reconstituted nucleosome cores demonstrated that they contain H2B, H3, H4 and HMG2 and are selectively deficient in H2A. The circular dichroism of DNA of the reconstituted cores was indistinguishable from that of control nucleosome cores. The results suggest that HMG2 replaces H2A as a component of the nucleosome histone core during reconstitution.

The kinetic studies of chemical modifications of the lysine and arginine residues of the nucleosome core particle from chicken erythrocytes with large molar excesses of 2,4,6-trinitrobenzenesulfonic acid and 2,3-butanedione, respectively, were performed over 20 mM-2.0 M NaCl. Each modification reaction was divided into the two, a rapid and an extremely slow, pseudo-first-order processes, and the numbers of the exposed and buried residues of the respective amino acids were determined. At very low ionic strength (20 mM boric buffer) all residues were inaccessible to the reagents. Between 0.3 and 0.6 M NaCl the basic amino acid residues in a nucleosome core particle were classified into the following three groups: (1) 42 arginine and 78 lysine residues in the N-terminal and C-terminal regions of the histones that are exposed to solvent, (2) a small number of arginine residues (approximately less than 14 Arg) that are strongly bound to the DNA phosphates, and (3) 48 arginine and 38 lysine residues buried in the globular region of the histone octamer. The results suggest that a small number of arginine residues play an essential role in the folding of DNA into a nucleosome core particle.

The nucleosome core binds more than two molecules of HMG17 at low ionic strength (8.9 mM Tris-HCl/8.9 mM boric acid/0.25 mM Na2EDTA, pH 8.3). Circular dichroism of the complexes showed only minor conformational changes of the nucleosome core DNA on binding of HMG17, with no detectable change in the histone secondary structure. The fluorescence of N-(3-pyrene) maleimide bound to -SH groups at Cys-110 of H3 histones in the core particle suggested that the structure of the histone octamer assembly changed little upon binding of HMG17 to the nucleosome. These observations support the idea that even a high level of HMG17 binding, e.g., four HMGs per nucleosome, alone, does not open up the core particle.

Comparative studies on the interaction of 8-anilino-1-naphthalenesulfonate (ANS) with polylysine and polyarginine have been made by equilibrium dialysis and fluorescence or circular dichroism measurements, to investigate the structural characteristics of the polypeptides. The results are summarized as follows: (i) ANS binds to either of the polypeptides primarily by electrostatic interaction while hydrophobic interaction partially facilitates the dye binding; both interactions are stronger in the polyarginine-dye binding than the polylysine-dye binding. (ii) The fluorescence of ANS is more intensified when the dye binds to polyarginine than to polylysine regardless of the value of r (number of bound dye per amino-acid residue) of polypeptide-dye complexes, although the intensification depends on the r value and becomes maximum at r = 0.25–0.35 for both cases. (iii) The binding of ANS to each polypeptide is cooperative at r < 0.4. (iv) The circular dichroism is more efficiently induced in the spectral region of ANS by binding to polyarginine than to polylysine. From these results, it was concluded that, compared to polylysine, polyarginine suffers some structural change by ANS binding into a more compact molecular configuration having some regularity with a lower dielectric environment.

We have succeeded in growing Saccharomyces cerevisiae (baker's yeast) on media containing 2H2O and isolating the core histones highly deuterated in the non-exchangeable positions. The deuterated histones obtained here are of great value for their possible widespread use for structural studies of chromatin.

Soluble complexes of poly-L-lysine and poly-L-arginine with DNA were prepared by a salt gradient dialysis and were investigated on stoichiometric aspect using a dye binding technique. It was found that only partial cationic residues on polyarginine are effective for occupying DNA phosphate groups, while all residues in polylysine can occupy.

Induced optical activities of acridine orange and actinomycin D bound to DNA-polylysine and DNA-polyarginine complexes were measured in order to obtain some information about the DNA conformation in the complexes. In the case of acridine orange, the anomalous circular dichroism (CD) spectra obtained at high molar ratios of the dye to DNA phosphate in the visible and ultraviolet regions, differed greatly from the spectrum of the dye bound to free DNA. The ultraviolet CD spectra of actinomycin D bound to the complexes also differed markedly from that of actinomycin D bound to free DNA. The CD spectra of DNA-polylysine-dye and DNA-polyarginine-dye systems were qualitatively different from each other for both dyes. From these findings, the possibility of specific conformational alterations of the free DNA portions in each DNA-polypeptide complex with the two basic polypeptides were suggested.

Circular dichroism spectra of acridine orange bound to E. coli tRNA were studied at varying tRNA phosphate-to-dye (P/D) ratios for both unfractionated and purified materials in the absence of Mg++. From the rather discrete features exhibited in the circular dichroism spectra three types of interactions were observed: (1) A high P/D ratio such as 75.2 or 49.8 indicates the interaction between the nucleotide base and dye molecule. The spectra with a large positive peak at 515 mμ are, however, quite different from that of DNA–AO complex under similar conditions. (2) With an intermediate P/D ratio (26.5 to 9.6) dye molecules bound strongly to the polynucleotide chain. (3) With low P/D ratios (≤7.5) the interaction appears to be due to the stacked dye molecules in the single-stranded part of tRNA. The spectra of the third group have an isobestic point at 477 mμ. Below a P/D ratio of 4 the spectrum shows one positive and two negative bands which may be the characteristics of circular dichroism of stacked dyes in polynucleotide chain. Although no drastic change in the conformation of tRNA itself was detectable in the presence of Mg++ in the ultraviolet region, a dramatic change was observed in the circular dichroism of tRNA–acridine orange complex when Mg++ concentration was increased to 10−3M. It was inferred that certain conformational changes other than simple hydrogen bond formation occured in tRNA molecules at this high Mg++ concentration, so that the amount of bound dye in the stacking condition was increased through the transition.