A re-examination of the crystal structure of A-DNA using fiber diffraction data

Classical A-DNA helices with h = 0.25 nm may represent the greatest mass per unit length attainable by polynucleotide duplexes. The X-ray diffraction pattern from polycrystalline and well-oriented fibers of calf thymus DNA in its A-form has been carefully re-examined. Indexing on the basis of a C-face-centered monoclinic unit cell of dimensions a = 2.170 nm, b = 3.990 nm, c = 2.803 nm and beta = 96.82 degrees is superior to alternatives that have been proposed. Two right-handed. Watson-Crick base-paired, helical DNA chains with 2 X 11 nucleotides per 2.803 nm pitch, each carrying C3'-endo furanose rings, pass through the unit cell. The crystallography requires the two chains in the duplex to be antiparallel and conformationally identical but the 11 nucleotides in each pitch may be distinct. However, a secondary structure with a mononucleotide asymmetric unit provides as good an X-ray agreement as one with 11 distinct nucleotides. This relative lack of variability is quite different from what is observed in fibrous B-DNAs.     

Evidence for allosteric transitions in secondary structure induced by superhelical stress

Previous studies suggest that the global secondary structures of native supercoiled and equilibrium linear DNAs may differ somewhat. Recent evidence also indicates that metastable secondary structure commonly persists following complete relaxation of the superhelical stress by intercalating dyes or by the action of topoisomerase I. In this work, the torsion constants (alpha) of pBR322, pUC8 and M13mp7 (replicative form) DNAs are determined by time-resolved fluorescence polarization anisotropy at various times subsequent to linearization. In all three cases, the torsion constants are relatively low immediately after linearization, and evolve for eight to ten weeks before reaching their apparent equilibrium values. It is shown in detail how the persistence of metastable secondary structure, subsequent to relaxation of superhelical stress, necessarily implies that one or more transitions in equilibrium secondary structure are induced as the superhelix density is varied from zero to native, or vice versa. Samples of pUC8 dimer (5434 base-pairs) with different superhelix densities are prepared by the action of topoisomerase I in the presence of various amounts of ethidium. Their median linking number differences are determined by standard band counting methods. The translational diffusion coefficient (Do) and the plateau diffusion coefficient (Dplat) characterizing internal motions over short distances (225 A) are determined by dynamic light-scattering. The torsion constant (alpha) between base-pairs and the circular dichroism spectrum are also measured for each sample. Curves of Dplat, Do, alpha and molar ellipticity ([theta]) (at the minimum near 250 nm) versus superhelix density (sigma) are constructed. The curve of Do versus sigma is very similar to that for sedimentation coefficient versus sigma for simian virus 40 (SV40) and polyoma DNAs. The curves of Dplat, Do, alpha and [theta] versus sigma show that, with increasing negative superhelix density, a structural transition occurs near sigma = -0.020 to an intermediate state with low torsion constant, and a second structural transition occurs near sigma = -0.035 to a state that exhibits more normal properties by sigma = -0.048. These data are consistent with the hypothesis that supercoiling induces two successive allosteric transitions to alternative global secondary structures. The data are much less consistent with the hypothesis that supercoiling induces some radical secondary structure at one or a few sites of small extent at sigma = -0.020, and at other sites at sigma = -0.035, or with hypotheses based on changes in tertiary structure alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Circular dichroism and DNA secondary structure.

The change in average rotation of the DNA helix has been determined for the transfer from 0.05 M NaCl to 3.0 M CsCl, 6.2 M LiCl and 5.4 M NH4Cl. This work, combined with data at lower salt from other laboratories, allows us to relate the intensity of the CD of DNA at 275 nm directly to the change in the number of base pairs per turn. The change in secondary structure for the transfer of DNA from 0.05 M NaCl (where it is presumably in the B-form) to high salt (where the characteristic CD has been interpreted as corresponding to C-form geometry) is found to be -0.22 (+/- 0.02) base pairs per turn. In the case of mononucleosomes, where the CD indicates the "C-form", the change in secondary structure (including temperature effects) would add -0.31 (+/- 0.03) turns about the histone core to the -1.25 turns estimated from work on SV40 chromatin. Accurate winding angles and molar extinction coefficients were determined for ethidium.     

Influence of embedding media on DNA structure in herpes simplex virus type 1

The organization of encapsidated herpes simplex viral DNA in situ was examined by use of the osmium-amine stain specific for DNA. After either formaldehyde or glutaraldehyde fixation the DNA is packaged in a compact toroid without inner structure with Epon or GMA embedment but revealed a complex inner structure with Lowicryl K4M embedment. In the latter there was an inner cylindrical core, 50 X 80 nm, around which were apposed one or more thick filaments of 5-8 nm diameter. Thinner DNA filaments of 3-4 nm diameter form a cage of loose coils around the core with an intervening space of approximately 15 nm. Lowicryl embedding may be considered as a tool to investigate the packaging of viral DNA in virions.     

Comparative vibrational spectroscopy of intracellular tau and extracellular collagen I reveals parallels of gelation and fibrillar structure

The N-terminal tau 2-19 peptide undergoes gelation, syneresis, and aggregation over a period of years. These changes may be approximated on a shorter time scale by agitation and partial dehydration. The anomalously enhanced (229 nm) ultraviolet resonance Raman (UVRR) imide II band reveals a common structural feature for gels of nondehydrated tau 2-19 and collagen I and insoluble paired helical filaments (PHFs) and collagen I of weak hydrogen bonding at proline carbonyls. Anomalous UVRR enhancement of amide bands at 229 nm results from gel structure, as demonstrated by increased amide absorption at the red edge for tau 2-19 gel and implies the involvement of water in gel structure. In aged, dehydrated tau 2-19 gel, proline carbonyls lose their bonds to water and tyrosine becomes deprotonated, as demonstrated by UVRR spectroscopy. The Fourier transform infrared (FTIR) amide I band shows that antiparallel beta-sheet structure increases with syneresis in the tau 2-19 hydrogel. The comparison of FTIR results for PHFs with collagen I gel and polyproline demonstrates that the secondary structure of PHFs is polyproline II. One implication of this assignment is that the fibrillation of hydrophilic tau is thermodynamically driven by the entropy gained as hydrogen-bonded water is freed, as for collagen I. The FTIR results also show that peptide domains culled from a longer protein do not necessarily fold into identical secondary structures. A pathological, sequential mechanism of gelation, syneresis, and fibrillation for tau in AD is suggested and is supported by the observation of amorphous neurofibrillary tangle development and fibrillation in vivo.     

Bovine lens crystallins do contain helical structure: a circular dichroism study.

In order to settle a recent discussion on the secondary structure of lens crystallins, we have measured the circular dichroism (CD) spectra of alpha-, beta(H)-, and beta(L)-crystallin from 178 to 250 nm and of gamma-crystallin from 168 to 250 nm. The results were analysed by means of a newly developed algorithm that almost doubles the reliability of secondary structure prediction and that allows discrimination between alpha- and 3(10)-helical, and between extended and polyproline beta-type structure. The results indicate that the crystallins studied contain a non-negligible amount of alpha-helical structure, although at least 50% of it is in the form of single and/or distorted loops. In alpha-crystallin, which is related to the chaperones, the helical content is lower than in beta- and gamma-crystallin. In some cases, the helices may play a role in DNA binding by the crystallins.     

Substrate and DNA binding to a 50-residue peptide fragment of DNA polymerase I. Comparison with the enzyme

The fluorescent nucleotide 2',3'-trinitrophenyl-ATP (TNP-ATP) binds at the triphosphate substrate binding site of the large (Klenow) fragment of DNA polymerase I (Pol I) as detected by direct binding studies measuring the increase in fluorescence of this ligand (n = 1.0, KD = 0.07 microM). The enzyme-TNP-ATP complex binds Mg2+ and Mn2+ tightly (KD = 0.05 microM) as measured by an increase in fluorescence on titrating with these metals. The substrate dGTP competitively displaces TNP-ATP from the enzyme (KD = 5.7 microM) de-enhancing the fluorescence. The polymerase reaction is half-maximally inhibited by 0.8 microM TNP-ATP in the presence of dATP (10 microM) as substrate. A region of the amino acid sequence of Pol I (peptide I) consisting of residues 728-777 has been synthesized and found to contain significant secondary structure by CD both in water and 50% methanol/water. In water at 3 degrees C, peptide I binds the substrate analog TNP-ATP (KD = 0.03 microM) with a stoichiometry of 0.2. In 50% methanol at 3 degrees C, peptide I binds TNP-ATP with a higher stoichiometry than in water, consistent with a 1:1 complex, but biphasically (16% of the peptide, KD = 0.09 microM; 84% of the peptide, KD = 5.0 microM), and competitively binds the Pol I substrates dATP, TTP, and dGTP (KD = 230-570 microM). Evidence from size exclusion high performance liquid chromatography suggests that these two forms of the peptide are monomer and dimer, respectively. Significantly, the peptide I-TNP-ATP complex binds duplex DNA, tightly (KD = 0.1-0.5 microM) and stoichiometrically, and single stranded DNA more weakly. The peptide I-duplex DNA complex binds both TNP-ATP (KD = 0.5-1.5 microM) and Pol I substrates (KD = 350-2100 microM) stoichiometrically. In a control experiment, a second peptide, peptide II, based on residues 840-888 of the Pol I sequence, retains secondary structure, as detected by CD, but displays no binding of TNP-ATP. The ability of peptide I, which represents only 8% of the large fragment of Pol I, to bind both substrates and duplex DNA indicates that residues 728-777 constitute a major portion of the substrate binding site of this enzyme.     

The effect of template secondary structure on vaccinia DNA polymerase.

Vaccinia virus DNA polymerase will utilize a substrate consisting of phi X174 DNA primed with a strand of a unique restriction fragment, but the reaction is inefficient. Examination of the reaction products by alkaline agarose gel electrophoresis revealed a few discrete fragments, each corresponding to an extended primer strand. This result implies that specific barriers exist on the phi X174 template which impede, but do not completely halt, the progress of the enzyme. Only a few per cent of the template molecules were completely copied. Similar findings were reported by Sherman and Gefter using Escherichia coli DNA polymerase II and fd DNA (J. Mol. Biol. (1976) 103, 61-76). Several observations suggest that the barriers are regions of template secondary structure. Some barriers are more effective than others, and they increase in both effectiveness and number as the temperature is decreased. The same barriers are observed with T4 DNA polymerase, but none are detected with E. coli DNA polymerase I. Finally, the major barriers are located in regions of the phi X174 sequence known to contain hairpin structures of relatively high stability. The exact stopping point of one of the major barriers is within the duplex stem of a hairpin structure. These results show that DNA polymerases are a useful probe of the secondary structure of a single-stranded DNA.     

Secondary structure determination in proteins from deep (192-223-nm) ultraviolet Raman spectroscopy

Raman intensities obtained with UV laser excitation at 223, 218, 204, 200, and 192 nm are reported for the amide I, II, III, and II' bands of random-coil polylysine. The excitation profiles show enhancement via the pi-pi electronic transition, at approximately 190 nm. Enhancement for amide I is weak, however, and most of the intensity can be accounted for by preresonance with a deeper UV transition at approximately 165 nm. The amide II' band dominates the spectrum in D2O, consistent with the suggestion that the main distortion coordinate in the pi-pi excited state is the stretching of the C-N peptide bond. Amide II intensities with 200- and 192-nm excitation are reported for several proteins. The previously reported negative linear correlation with alpha-helix content (due to Raman hypochromism in the alpha-helices) is found not to apply to proteins with high beta-sheet content when the excitation wavelength is 200 nm. Much higher intensities are seen for these proteins and are attributed to a red shift of the pi-pi absorption for the beta-structure. A linear correlation with alpha-helix content is found for excitation of 192 nm, which corresponds to an isosbestic point of the beta-sheet and random-coil absorption bands. Characteristic amide II Raman cross sections are derived for alpha-helical, beta-sheet, and random-coil elements and are used to determine secondary structure for alpha 1- and beta-purothionin, by use of amide II intensities with 200- and 192-nm excitation. The results are in good agreement with a previous determination based on amide I band deconvolution in off-resonance Raman spectra.     

Chromosomal structure of the halophilic archaebacterium Halobacterium salinarium.

The chromosomal structure of the extremely halophilic archaebacterium Halobacterium salinarium was examined. Sheared chromosomes prepared from the bacteria in the late exponential phase were separated into two peaks (peaks I and II) by sucrose gradient centrifugation, suggesting that the chromosomes consist of two parts differing in quality. The UV spectra of peaks I and II resembled those of DNA and eukaryotic chromatin, respectively. Electron microscopic observations revealed that the major component of peak I was protein-free DNA, while the major components of peak II were rugged thick fibers with a diameter of 17 to 20 nm. The rugged fibers basically consisted of bacterial nucleosome-like structures composed of DNA and protein, as demonstrated in experiments with proteinase and nuclease digestion. Whole-mount electron microscopic observations of the chromosomes directly spread onto a water surface revealed a configuration in which the above-described regions were localized on a continuous DNA fiber. From these results it is concluded that the H. salinarium chromosome is composed of regions of protein-free DNA and DNA associated with nucleosome-like structures. Peaks I and II were predominant in the early exponential phase and stationary phase, respectively; therefore, the transition of the chromosome structure between non-protein-associated and protein-associated forms seems to be related to the bacterial growth phase.     

Binding of tachyplesin I to DNA revealed by footprinting analysis: significant contribution of secondary structure to DNA binding and implication for biological action.

In view of the cationic amphipathic structure of tachyplesin I and antiparallel beta-sheet as a general DNA binding motif, DNA binding of the antimicrobial peptide has been examined. Several footprinting-like techniques using DNase I protection, dimethyl sulfate protection, and bleomycin- (BLM-) induced DNA cleavage were applied in this study. Some distinct footprints with DNase I are detected, and also the sequence-specific cleavage mode of the BLM-Fe(II) complex clearly is altered in the presence of tachyplesin I. In addition, methylation of the N-7 residue of guanine situated in the DNA major groove is not entirely inhibited (or activated) by tachyplesin I. The results suggest that tachyplesin I interacts with the minor groove of DNA duplex. Disappearance of the footprints by dithiothreitol-treated tachyplesin I and Ala-tachyplesin strongly suggests a significant contribution of secondary structure containing an antiparallel beta-sheet to the DNA binding of tachyplesin I. This is the first report on DNA interaction with a small peptide which contains a unique antiparallel beta-sheet structure. The mechanism for antimicrobial action of tachyplesin I has also been inferred.     

Predicting the three-dimensional structure of alpha- and beta-interferons

Secondary structures of leukocyte alpha 1- and alpha 2-interferons and of fibroblast beta-interferon are calculated using the molecular theory of protein secondary structures. The common secondary structure calculated for alpha- and beta-interferons is used to predict the three-dimensional structures of fragments 1-110 and 111-166 of the chains (which are supposed to be quasi-independent domains). The predicted structure of the active domain I (1-110) is an "up-and-down" tetrahelical complex (in which the second helix is shorter than the others and can be missed in alpha 1-interferon) similar to the mirror-image of myohaemoerythrin. The predicted structure of domain II (111-166) is either a three-stranded beta-sheet screened from one side by two alpha-helices or a three-helical complex (similar to that in the N-domain of papain), the first structure being better consistent with the circular dichroism data of alpha-interferon and its C-end fragment.     

Secondary structure in properdin of the complement cascade and related proteins: a study by Fourier transform infrared spectroscopy

Six structural repeat motifs of 58 amino acids are found in the sequence of both mouse and human properdins. Twelve more examples of the motif are available from the sequences of thrombospondin, the terminal complement components, and the thrombospondin-related anonymous protein. The averaged Robson and Chou-Fasman secondary structure predictions show that there are 57-66% turn and 19-38% beta-sheet structures in the typical repeat motif. The high amount of turn structure is consistent with Gly, Pro, Cys, and Ser being the four most abundant amino acid residues in properdin. Comparisons with sequences found in the circumsporozoite protein from several species of malaria parasites show that their sequences and secondary structures strongly coincide only in a 18-residue segment. Further secondary structure analysis utilized Fourier transform infrared spectroscopy of human properdin in 2H2O buffers. These show a broad amide I band that, after second-derivative and deconvolution calculations, is shown to be composed of several components. Two at 1633 and 1683 cm-1 are strong evidence for beta-sheet structure, although overlap from beta-turns can also contribute. The presence of beta-turn structure is indicated by absorptions at 1662-1675 and 1645 cm-1. The properdin structure contains substantial quantities of beta-sheet and beta-turn structures, which is consistent with the secondary structure predictions and amino acid compositions. The length of the repeat motif is estimated as 3.3-4.3 nm, and an estimated 14-22% of nonexchanged amide protons reside in properdin. This is suggestive of a high degree of solvent accessibility in the structure.     

A comparative study on the interaction of acridine and synthetic bis-acridine with G-quadruplex structure

DNA from the telomeres contains a stretch of simple tandemly repeated sequences in which clusters of G residues alternate with clusters of T/A sequences along one DNA strand. Model telomeric G-clusters form four-stranded structures in presence of Na(I), K(I) and NH(4)(I) ions. Electrophoretic and spectroscopic studies were made with the telomeric related sequences d(T6G16) or d(G4T2G4T2G4T2G4). It was noticed earlier that G-quadruplex may either be inter-molecular, or intra-molecular, or a mixture of both. CD spectral characteristics of various G-quadruplex DNA suggests that the CD maximum at 293 nm corresponds to that of an intra-molecular G-quadruplex structure or hairpin dimers. Fluorescence titration studies also show that acridine and the bis-acridine are interacting with G-quadruplex DNA and destabilize the K(I)-quadruplex structure more efficiently than the quadruplex formed by NH(4)(I) ion. Among the two drugs studied, acridine is more capable of breaking the G-quadruplex structure than bis-acridine. This result is further confirmed by the CD experiments.     

Determination of secondary structure in rabbit globin messenger RNA by thermal denaturation.

The secondary structure of highly purified globin messenger RNA has been investigated by alkaline hydrolysis, nuclease digestion, and thermal denaturation. The thermal denaturation properties of globin messenger have been compared to poly(U), poly (A), and a synthetic random sequence RNA copolymer. From these studies it is concluded that globin mRNA contains considerable secondary structure and that the amount of helical structure is greater than that which occurs with a random sequence polyribonucleotide. Globin mRNA contains, by comparison to the secondary structures of native DNA, tRNAs, or 18S rRNA, helices with involve 55-62% of the bases or 58-68% if a correction is made for the 3'-terminal poly(A) segment. The helices of globin mRNA appear to be unique as differences in the NaCl stabilization of this RNA have been noted when compared to other naturally ooccurring and synthetic RNAs. Comparison of the hyperchromicity maxima, obtained at 260 and 280 nm for globin mRNA and 18S rRNA, indicates that the helices of the two RNAs contain similar numbers of G-C base pairs. Differential analysis of NaCl stabilization curves indicate three discrete thermally denaturable helix types in globin mRNA.     

Phylogenetic analysis and secondary structure of the Bacillus subtilis bacteriophage RNA required for DNA packaging

An unusual RNA molecule encoded by the Bacillus subtilis bacteriophage phi 29 is a structural component of the viral prohead and is required for the ATP-dependent packaging of DNA. Here we report a model of secondary structure for this prohead RNA developed from a phylogenetic analysis of the primary sequences of prohead RNAs of related phages. Twenty-nine phages related to phi 29 were found to produce prohead RNAs. These RNAs were analyzed by their ability to replace phi 29 RNA in in vitro phage assembly, by Northern blot hybridization with a probe complementary to phi 29 RNA, and by partial and complete sequence analyses. These analyses revealed four quite different sequences ranging in length from 161 to 174 residues. The secondary structure deduced from these sequences, in agreement with earlier observations, indicated that prohead RNA is organized into two domains. The larger 5'-domain (Domain I) is composed of 113-117 residues and contains four helices. Three of these helices appear to be organized into a central stem that is interrupted by two unpaired loops and the fourth helix and loop. The smaller 3'-domain (Domain II) is composed of 40-44 residues and consists of two helices. Domains I and II are separated by 8-13 unpaired residues. Nuclease cleavage occurs readily in this single-stranded joining region, and this cleavage allows the subsequent separation of the two RNA domains. The separated Domain I is fully active in DNA packaging in vitro. The functional significance and biological role of Domain II are unknown. The phylogenetic secondary structure model provides a basis for further analysis of the role of this RNA in bacteriophage morphogenesis.     

Introduction of interrupted secondary structure in supercoiled DNA as a function of superhelix density: consideration of hairpin structures in superhelical DNA.

PM2 DNA was prepared with different superhelical densities (sigma) in order to examine the relationship betweenn supercoiling and the occurrence of a region(s) of unpaired bases in this DNA. A previous study showed that CH3HgOH reacts with native superhelical PM2 DNA more rapidly than the nicked form II. This evaluation of binding, monitored through the change of sedimentation velocity, was repeated on PM2 DNA I with different superhelical densities. Early binding is detected by an increase in sedimentation velocity and occurs with molecules with sigma' values betwee -0.025 and -0.037. The conversion of form I to form II with the single-strand-specific endonuclease from Neurospora crassa also occurs above a sigma value of -0.025. This data strongly supports the view that supercoiling produces interrupted secondary structure. The question whether the interrupted regions remain single stranded in character or form small intrastrand hairpin regions is considered by examining which model best fits the CH3HgOH- induced sedimentation velocity changes and the standard sedimentation velocity versus the superhelical density curve for the in vitro made DNAs. The hairpin model offers the most satisfactory explanations for all the results of this and previous studies.     

The unfolding of our understanding of RNA structure: a personal reflection

In this article, I review how our research on RNA began, how it led us to demonstrate the single-stranded nature of RNA, and the ways in which it differs from double-stranded DNA. It was based on the development of a method for the isolation of undegraded rRNA and the observation that in rRNA preparations due to their viscosity behavior resemble a flexible, contractile coil. In support of this assumption, birefringence of flow measurements showed that rRNA solutions gave moderate positive values, which disappeared upon addition of salt. This is in contrast with DNA solutions where considerable negative birefringence persists even in the presence of salt. Further studies on RNA showed a close correlation of the ionic strength dependencies of optical rotation, optical density and hydrodynamic properties. These early results indicated that rRNA and tRNA possess a significant secondary structure. I then review the basis of the hairpin model for the secondary structure of RNA and finally, summarize current understanding of the tertiary structure of RNA.     

Nucleosome structure.

Electron microscopic and biochemical results are presented supporting the following conclusions: (1) Two molecules of each histone H2A, H2B, H3 and H4 are necessary and sufficient to form a nucleosome with a diameter of 12.5 +/- 1 nm and containing about 200 base pairs of DNA. (2) H3 plus H4 alone can compact 129 +/- 8 DNA base pairs into a sub-nucleosomal particle with a diameter of 8 +/- 1 nm. In such a particle the DNA duplex is under a constraint equivalent to negative superhelicity. (3) Chromatin should be viewed as a dynamic structure, oscillating between a compact structure (the nucleosome) and more open structures, depending on the environmental conditions.     

Emergence of polyproline II-like structure at early stages of experimental evolution from random polypeptides

To examine whether a primordial functional protein at the early stages of evolution has structural features, we carried out experimental evolution consisting of 25 cycles (generations) of mutation and selection toward DNA-binding function using a random-sequence polypeptide of 139 amino acid residues with no secondary structure as the initial sequence. In each generation, 16 clones were sampled arbitrarily for sequence analysis, and a phylogenetic tree was constructed. Polypeptide evolution proceeded from the initial point on branch I in 2 main directions of branches II and III. The initial and 2 evolved polypeptides (one at the 24th generation on branch III and the other at the 25th generation on branch II) were purified to examine their functional and structural properties. Although binding of the initial polypeptide to the target DNA was not detected by surface plasmon resonance measurements, the 2 evolved polypeptides bound to the DNA with dissociation constants of 1.6 and 1.0 microM, respectively, indicating an increase in affinity during the experimental evolution. Circular dichroism spectra of the evolved polypeptides, but not of the initial polypeptide, showed features characteristic of the polyproline II (PPII)-like structure, a left-handed helical structure commonly found in natural proteins, suggesting that the structure emerged through the experimental evolution. The same structural feature was found in another experimental evolution toward catalytic activity. These results demonstrate that the PPII-like structure is one of the common features that could have appeared in the early evolutionary stages of primordial functional protein.