Interaction of rat testis protein, TP, with nucleic acids in vitro. Fluorescence quenching, UV absorption, and thermal denaturation studies

The nucleic acid binding properties of the testis protein, TP, were studied with the help of physical techniques, namely, fluorescence quenching, UV difference absorption spectroscopy, and thermal melting. Results of quenching of tyrosine fluorescence of TP upon its binding to double-stranded and denatured rat liver nucleosome core DNA and poly(rA) suggest that the tyrosine residues of TP interact/intercalate with the bases of these nucleic acids. From the fluorescence quenching data, obtained at 50 mM NaCl concentration, the apparent association constants for binding of TP to native and denatured DNA and poly(rA) were calculated to be 4.4 X 10(3) M-1, 2.86 X 10(4) M-1, and 8.5 X 10(4) M-1, respectively. UV difference absorption spectra upon TP binding to poly(rA) and rat liver core DNA showed a TP-induced hyperchromicity at 260 nm which is suggestive of local melting of poly(rA) and DNA. The results from thermal melting studies of binding of TP to calf thymus DNA at 1 mM NaCl as well as 50 mM NaCl showed that although at 1 mM NaCl TP brings about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From these results it is concluded that TP, having a higher affinity for single-stranded nucleic acids, destabilizes double-stranded DNA, thus behaving like a DNA-melting protein.     

Interaction between acridine orange and polyriboadenylic acid

The absorption spectra and circular dichroism of aqueous solutions of acridine orange mixed with polY(riboadenylic acid) [poly(rA)] have been measured for different mixing ratios at acid and neutral pH. The binding ratio of dye to poly(rA) has been determined by equilibrium dialysis. At acid pH where poly(rA) is in a double-stranded helix, monomeric dye molecules are intercalated between base pairs, first sparsely and then at neighbouring sites with mutual coupling, as the nucleotide-to-dye mixing ratio decreases. In the presence of excess dye, dimeric dye molecules of antiparallel type are bound to phosphate groups electrostatically and stack together to form linear sequences along a poly(rA) chain. At neutral pH where poly(rA) is single-stranded, isolated intercalation of monomeric dye molecules can occur in the helical parts. At intermediate mixing ratios, half-intercalated dimeric dye molecules are bound to adjacent sites and electronically coupled, inducing characteristic circular dichroism. In the presence of higher amounts of dye, external stacking of dimeric dye molecules of antiparallel type occurs along a poly(rA) chain. The binding of dye cations is suppressed to some degree at acid pH compared to that at neutral pH, owing to the repulsion exerted by protonated adenine bases.     

Induced circular dichroism in nucleic acid-acridine derivative complexes

Visible absorption and circular dichroism (CD) spectra have been measured for complexes formed between nucleic acids (calf thymus DNA, poly(rA).poly(rU) and poly(rI).poly(rC)) and 9-aminoacridines (quinacrine, acranil and 9-amino-6-chloro-2-methoxy acridine). With poly(rA).poly(rU), a new absorption band was observed at longer wavelengths. The nucleic acid-drug complexes showed considerable different induced CD spectra. Analysis of these CD spectra suggests that the cationic side chains of quinacrine and acranil play an important role on the binding properties to DNA and poly(rA).poly(rU).     

Complex formation between the adenovirus DNA-binding protein and single-stranded poly(rA). Cooperativity and salt dependence

The complex formed between adenovirus DNA-binding protein (AdDBP) and poly(rA) was investigated with circular dichroism spectroscopy. The binding process was studied at a variety of salt concentrations, and the titration curves were analyzed according to the contiguous cooperative binding model given by McGhee and von Hippel [McGhee, J.D., & von Hippel, P.H. (1974) J. Mol. Biol. 86, 469-489]. The cooperativity factor omega of the binding process is low (omega approximately 20-30) and independent of the salt concentration. This in contrast to the binding constant K for which a moderately strong salt dependence is observed: delta log (K omega)/delta log [NaCl] = -3.1. The size of the binding site was consistently calculated to be about 13. We also studied the C-terminal 39-kDa fragment which is sufficient for DNA replication in vitro. Complex formation between this fragment of AdDBP and poly(rA) appeared to be characterized by spectroscopic and binding properties similar to those of the intact protein. Only, the binding constant in 50 mM NaCl is a factor of 5 lower.     

Study of the binding of single-stranded DNA-binding protein to DNA and poly(rA) using electric field induced birefringence and circular dichroism spectroscopy

Binding of the single-stranded DNA-binding protein (SSB) of Escherichia coli to single-stranded (ss) polynucleotides produces characteristic changes in the absorbance (OD) and circular dichroism (CD) spectra of the polynucleotides. By use of these techniques, complexes of SSB protein and poly(rA) were shown to display two of the binding modes reported by Lohman and Overman [Lohman, T.M., & Overman, L. (1985) J. Biol. Chem. 260, 3594-3603]. The circular dichroism spectra of the "low salt" (10 mM NaCl) and "high salt" (greater than 50 mM NaCl) binding mode are similar in shape, but not in intensity. SSB binding to poly(rA) yields a complexed CD spectrum that shares several characteristics with the spectra obtained for the binding of AdDBP, GP32, and gene V protein to poly(rA). We therefore propose that the local structure of the SSB-poly(rA) complex is comparable to the structures proposed for the complexes of these three-stranded DNA-binding proteins with DNA (and RNA) and independent of the SSB-binding mode. Electric field induced birefringence experiments were used to show that the projected base-base distance of the complex is about 0.23 nm, in agreement with electron microscopy results. Nevertheless, the local distance between the successive bases in the complex will be quite large, due to the coiling of the DNA around the SSB tetramer, thus partly explaining the observed CD changes induced upon complexation with single-stranded DNA and RNA.     

Complex of fd gene 5 protein and double-stranded RNA

We report the formation of complexes of the single-stranded DNA binding protein encoded by gene 5 of fd virus, with natural double-stranded RNAs. In the first direct visualization of a complex of the fd gene 5 protein with a double-stranded nucleic acid, we show by electron microscopy that the double-stranded RNA complex has a structure which is distinct from that of complexes with single-stranded DNA and is consistent with uniform coating of the exterior of the double-stranded RNA helix by the protein. Circular dichroism spectral data demonstrate that the RNA double helix in the complex is undisrupted, and that perturbation of the 228-nm circular dichroism assigned to protein tyrosines can occur in the absence of intercalation of nucleotide bases with protein aromatic residues. Our findings emphasize the potential importance of interaction with the sugar-phosphate polynucleotide backbone in binding of the fd gene 5 protein to nucleic acids.     

Interaction of trivaline with single-stranded polyribonucleotides]

Binding of tripeptide H-Val3-(NH)2-Dns (TVP) to polyribonucleotides was studied by fluorescence methods, circular and flow linear dichroism, equilibrium dialysis and electron microscopy. It was found that TVP binds to poly(U) in monomer, dimer and tetramer forms with binding constants of about 10(3), 40, 18.10(4) M, respectively. The cooperativity parameter for peptide dimer binding is 2000. The peptide forms tetramer complexes with poly(A), poly(C), poly(G) also. The formation of a complex between the peptide tetramer and nucleic acid is accompanied by a significant increase in the fluorescence intensity. The cooperative binding of TVP dimers to poly(U), poly(A), poly(C) is accompanied by a dramatic decrease in the flexibility of polynucleotide chains. However, it has a small effect (if any) on the flexibility of the poly(G) chain. The observed similarity of thermodynamic, optical and hydrodynamic++ properties of TVP complexes with single-stranded and double-stranded nucleic acids may reflect a similarity in the geometries of peptide complexes with nucleic acids. Electron microscopy studies show that peptide binding to poly(U) and dsDNA leads to compactization of the nucleic acids caused by interaction between the peptide tetramers bound to a nucleic acid. At the first stage of the compactization process the well-organized rod-like particles are formed, each consisting of one or more single-stranded polynucleotide fibers. Increasing the peptide concentration stimulates a side-by-side association and folding of the rods with the formation of macromolecular "leech-like" structures with the thickness of 20-50 nm.     

Salt-dependent binding of Escherichia coli initiation factor 3 to nucleic acids determined by sedimentation partition chromatography

The binding of 14CH3- initiation factor 3 (IF3) to polynucleotides is strongly dependent upon the concentration of added salt. The observed association constant, Kobs, increases by ca. a factor of 10(2) when the NaCl concentration is lowered from 200 to 100 mM for the binding of 14CH3-IF3 to all nucleic acids examined. This salt-dependent binding suggests that at physiological salt concentrations the formation of an IF3-polynucleotide complex is primarily driven by the release of cations from the nucleic acid, although anion effects are involved also. For single-stranded nucleic acids, nonelectrostatic interactions may contribute a factor of 10(2) to the value of Kobs, although accurate assessment of these interactions is complicated by anion effects. The binding of 14CH3-IF3 to the double helix, poly(A).poly(U), appears to be exclusively electrostatic. 14CH3-IF3 forms a maximum of 8 +/- 2 ion pairs with most single-stranded polynucleotides. The value of Kobs increases from ca. 10(3) to 10(5) M-1 when the NaCl concentration is lowered from 200 to 100 mM for the binding of 14CH3-IF3 to poly(A), poly(C), poly(U), and poly(A).poly(U). At physiological salt concentrations, IF3 shows no preference for any of these bases or for single or double-stranded structures. However, 14CH3-IF3 binds ca. 60 times greater to poly(A,G), at al NaCl concentrations examined, than to the other nucleic acids, indicating that IF3 has some preference for guanine-containing polynucleotides. The presence of 10 mM Mg2+ tends to reduce the value of Kobs at any given NaCl concentration, but to a smaller degree than predicted by simply a competition between Mg2+ and IF3 for the nucleic acid lattice.     

Nucleolin promotes secondary structure in ribosomal RNA

The effect of nucleolin on the secondary structure of RNA was studied using circular dichroism (CD). Nucleolin caused decreases in the main positive bands and shifts to higher wavelengths in the CD spectra of synthetic polynucleotides such as poly(G) and poly(A) indicating helix destabilizing activity. In contrast, nucleolin effected increases in signal and shifts to lower wavelengths of the peaks of CD spectra of ribosomal RNA, suggesting enhancement of secondary structure. Another major nucleolar RNA binding protein, B23, had helix destabilizing activity but did not enhance RNA secondary structure. It is proposed that nucleolin promotes formation of secondary structure in preribosomal RNA during the early stages of ribosome biogenesis.     

Purification and characterization of the coliphage N4-coded single-stranded DNA binding protein

We have purified and characterized a single-stranded DNA binding protein (N4 SSB) induced after coliphage N4 infection. It has a monomeric molecular weight of 31,000 and contains 10 tyrosine and 1-2 tryptophan amino acid residues. Its fluorescence spectrum is dominated by the tyrosine residues, and their fluorescence is quenched when the protein binds single-stranded DNA. Fluorescence quenching was used as an assay to quantitate binding of the protein to single-stranded nucleotides. The N4 single-stranded DNA binding protein binds cooperatively to single-stranded nucleic acids and binds single-stranded DNA more tightly than RNA. The binding involves displacement of cations from the DNA and anions from the protein. The apparent binding affinity is very salt-dependent, decreasing as much as 1,000-fold for a 10-fold increase in NaCl concentration. The degree of cooperativity (omega) is relatively independent of salt concentration. At 37 degrees C in 0.22 M NaCl, the protein has an intrinsic binding constant for M13 viral DNA of 3.8 x 10(4) M-1, a cooperativity factor omega of 300, and binding site size of 11 nucleotides per monomer. The protein lowers the melting point of poly(dA.dT).poly(dA-dT) by greater than 60 degrees C but cannot lower the melting transition or assist in the renaturation of natural DNA. N4 single-stranded DNA binding protein enhances the rate of DNA synthesis catalyzed by the N4 DNA polymerase by increasing the processivity of the N4 DNA polymerase and melting out hairpin structures that block polymerization.     

Phase equilibrium in poly(rA).poly(rU) complexes with Cd2+ and Mg2+ ions, studied by ultraviolet, infrared, and vibrational circular dichroism spectroscopy

Ultraviolet (UV) and infrared (IR) absorption and vibrational circular dichroism (VCD) spectroscopy were used to study conformational transitions in the double-stranded poly(rA). poly(rU) and its components-single-stranded poly(rA) and poly(rU) in buffer solution (pH 6.5) with 0.1M Na+ and different Mg2+ and Cd2+ (10(-6) to 10(-2) M) concentrations. Transitions were induced by elevated temperature that changed from 10 up to 96 degrees C. IR absorption and VCD spectra in the base-stretching region were obtained for duplex, triplex, and single-stranded forms of poly(rA) . poly(rU) at [Mg2+],[Cd2+]/[P] = 0.3. For single-stranded polynucleotides, the kind of conformational transition (ordering --> disordering --> compaction, aggregation) is conditioned by the dominating type of Me2+-polymer complex that in turn depends on the ion concentration range. The phase diagram obtained for poly(rA) . poly(rU) has a triple point ([Cd2+] approximately 10(-4)M) at which the helix-coil (2 --> 1) transition is replaced with a disproportion transition 2AU --> A2U + poly(rA) (2 --> 3) and the subsequent destruction of the triple helix (3 --> 1). The 2 --> 1 transitions occur in the narrow temperature interval of 2 degrees -5 degrees . Unlike 2 --> 1 and 3 --> 1 melting, the disproportion 2 --> 3 transition is a slightly cooperative one and observed over a wide temperature range. At [Me2+] approximately 10(-3) M, the temperature interval of A2U stability is not less than 20 degrees C. In the case of Cd2+, it increases with the rise of ion concentration due to the decrease of T(m) (2-->3). The T(m) (3-->1) value is practically unchanged up to [Cd2+] approximately 10(-3)M. Differences between diagrams for Mg(2+) and Cd2+ result from the various kinds of ion binding to poly(rA).poly-(rU) and poly(rA).     

Orientation of the bases of single-stranded DNA and polynucleotides in complexes formed with the gene 32 protein of bacteriophage T4. A linear dichroism study

Linear dichroism measurements were performed in the wavelength region 250 to 350 nm on complexes between the single-stranded DNA binding protein of bacteriophage T4 (gp32) and single-stranded DNA and a variety of homopolynucleotides in compressed polyacrylamide gels. The complexes appeared to orient well, giving rise to linear dichroism spectra that showed contributions from both the protein aromatic residues and the bases of the polynucleotides. In most cases the protein contribution appeared to be very similar, and the linear dichroism of the bases could be explained by similar orientations of the bases for most of the complexes. Assuming a similar, regular structure for most of the polynucleotides in complex, only a limited set of combinations of tilt and twist angles can explain the linear dichroism spectra. These values of tilt and twist are close to (-40 degrees, 30 degrees), (-40 degrees, 150 degrees), (40 degrees, -30 degrees) or (40 degrees, -150 degrees), with an uncertainty in both angles of about 15 degrees. Although the linear dichroism results do not allow a choice between these possible orientations, the latter two combinations are not in agreement with earlier circular dichroism calculations. For the complexes formed with poly(rC) and poly(rA), the linear dichroism spectra could not be explained by the same base orientations. In these two cases also the protein contribution to the linear dichroism appeared to be different, indicating that for some aromatic residues the orientations are not the same as those in the other complexes. The different structures of these complexes are possibly related to the relatively low binding affinity of gp32 to poly(rC), and to a lesser extent to poly(rA).     

Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids

Characterization of mammalian heterogeneous nuclear ribonucleoprotein complex protein A1 is reported after large-scale overproduction of the protein in Escherichia coli and purification to homogeneity. A1 is a single-stranded nucleic acid binding protein of 320 amino acids and 34,214 Da. The protein has two domains. The NH2-terminal domain is globular, whereas the COOH-terminal domain of about 120 amino acids has low probability of alpha-helix structure and is glycinerich. Nucleic acid binding properties of recombinant A1 were compared with those of recombinant and natural proteins corresponding to the NH2-terminal domain. A1 bound to single-stranded DNA-cellulose with higher affinity than the NH2-terminal domain peptides. Protein-induced fluorescence enhancement was used to measure equilibrium binding properties of the proteins. A1 binding to poly (ethenoadenylate) was cooperative with the intrinsic association constant of 1.5 X 10(5) M-1 at 0.4 M NaCl and a cooperativity parameter of 30. The NH2-terminal domain peptides bound noncooperatively and with a much lower association constant. With these peptides and with intact A1, binding was fully reversed by increasing [NaCl]; yet. A1 binding was much less salt-sensitive than binding by the NH2-terminal domain peptides. A synthetic polypeptide analog of the COOH-terminal domain was prepared and was found to bind tightly to poly-(ethenoadenylate). The results are consistent with the idea that the COOH-terminal domain contributes to A1 binding through both cooperative protein-protein interaction and direct interaction with the nucleic acid.     

The in vitro DNA-binding properties of purified nuclear lamin proteins and vimentin

The ability of purified nuclear lamin A, lamin B, lamin C, and vimentin from Ehrlich ascites tumor cells to bind nucleic acids was investigated in vitro via a quantitative filter binding assay. At low ionic strength, vimentin bound more nucleic acid than the nuclear lamins and showed a preference for G-containing nucleic acids. Nuclear lamins A and C were quite similar in their binding properties and bound G- and C-containing nucleic acids preferentially. The binding of poly(dT) by the lamins A and C was reduced in competition experiments by both poly(dG) and poly(dC), but not by poly(dA). Lamin B bound only oligo and poly(dG); no other nucleic acids tested were bound or could compete with the binding of oligo(dG). Vimentin, lamin A, and lamin C specifically bound a synthetic oligonucleotide human (vertebrate) telomere model. The Ka for vimentin (2.7 X 10(7) M-1) was approximately 10-fold higher than those for lamin A (2.8 X 10(6) M-1) and lamin C (2.9 X 10(6) M-1). Lamin B did not bind detectable amounts of the telomere model. Washing of lamin A- and lamin C-nucleic acid complexes, formed at low ionic strength, with solutions containing 150 mM KCl resulted in the elution of 30% of bound poly(dG)12-18 and 70% of bound synthetic oligonucleotide telomere model. These results, using purified individual proteins, are in good agreement with data from competition experiments with vimentin but are at odds with data obtained previously using a crude preparation of nuclear matrix proteins containing all three nuclear lamin proteins (Comings, D. E., and Wallack, A. S. (1978) J. Cell Sci. 34, 233-246). The nuclear lamins A and C and vimentin possess nucleic acid-binding properties that might permit their binding to specific base sequences and/or unique DNA structure, such as that observed for the binding of the telomere model. The significance of the higher affinity binding of nucleic acids by the cytoplasmic protein vimentin (compared with the nuclear lamins) remains to be elucidated.     

Hydrodynamic studies of a DNA-protein complex. Elongation of single stranded nucleic acids upon complexation with the gene 32 protein of phage T4 deduced from electric field-induced birefringence experiments

Short DNA and RNA fragments complexed with the helix destabilizing protein of bacteriophage T4, GP32, have been studied in solution by electric birefringence and circular dichroism. The birefringence of the complexes is positive and the magnitude indicates that the DNA and RNA fragments become linear and rigid upon protein binding. The field free decay is biphasic. On the basis of a rigid rod approximation the slow relaxation time leads to a base-base distance along the helix axis in the complex from 4.3 to 5.6 A, an elongation of at least 50% compared to single-stranded DNA.     

Poly(rA) binds poly(rG).poly(rC) to form a triple helix.

Poly(rA) binds poly(rG).poly(rC) to form a triple helix. Evidence for this structure includes ultraviolet absorbance mixing curves and melting curves, and circular dichroism spectroscopy. The formation of the triple helix depends on the length of the poly(rC) strand. Triple helix forms when the average length is around 100 nucleotides but does not form when the average length is about 500 nucleotides.     

Physical studies of the interaction of a calf thymus helix-destablizing protein with nucleic acids

UP1, a calf thymus protein that destabilizes both DNA and RNA helices, dramatically accelerates the conversion of the inactive conformers of several small RNA molecules to their biologically active forms [Karpel, R. L., Swistel, D. G., Miller, N. S., Geroch, M. E., Lu, C., & Fresco, J. R. (1974) Brookhaven Symp. Biol. 26, 165-174]. Using circular dichroic and spectrophotometric methods, we have studied the interaction of this protein with a variety of synthetic polynucleotides and yeast tRNA3Leu. As judged by perturbations in polynucleotide ellipticity or ultraviolet absorbance, the secondary structures of the single-stranded helices poly(A) and poly(C), as well as the double-stranded helices poly[d(A-T)] and poly(U.U), are largely destroyed upon interaction with UP1 at low ionic strength. This effect can be reversed by an increase in [Na+]: half the UP1-induced perturbation of the poly(A) CD spectrum is removed at 0.05 M Na+. The variation of poly(A) ellipticity and ultraviolet absorbance with [UP1]/[poly(A)]p is used to determine the length of single-stranded polynucleotide chain covered by the protein: 7 +/- 1 residues. A model is presented in which the specificity of UP1 for single strands and their concomitant distortion are a consequence of maximal binding of nucleic acid phosphates to a unique matrix of basic residues on the protein. Analogous to the effect on polynucleotides, UP1-facilitated renaturation of yeast tRNA3Leu follows the partial destruction of the inactive tRNA's secondary structure. At the tRNA absorbance maximum, UP1 effects a hyperchromic change of 10%, representing one-third of the secondary structure of the inactive conformer. This change is also clearly observable as a perturbation of the tRNA's circular dichroism spectrum.     

The LexA repressor and its isolated amino-terminal domain interact cooperatively with poly[d(A-T)], a contiguous pseudo-operator, but not with random DNA: a circular dichroism study

The interaction of the entire LexA repressor and its amino-terminal DNA binding domain with poly[d(A-T)] and random DNA has been studied by circular dichroism. Binding of both protein species induces an about 2-fold increase of the positive circular dichroism band at about 270 nm of both polynucleotides, allowing a precise determination of the principal parameters as a function of mono- and divalent salt concentration and pH. Both proteins interact much more strongly (about 2000-fold) with poly[d(A-T)] than with random DNA as expected from the homology with the specific consensus binding site of LexA (CTGTATATATATACAG). For both LexA and its DNA binding domain we find that the interaction with poly[d(A-T)] is cooperative with a cooperativity factor omega of about 50-70 for both proteins over a wide range of solvent conditions, suggesting that the carboxy-terminal domain of LexA is not involved in this type of cooperativity. On the contrary, no cooperativity could be detected for the interaction of the LexA DNA binding domain with a random DNA fragment. The overall binding constant K omega (or simply K in the case of random DNA) depends strongly on the salt concentration as observed for most protein-DNA interactions, but the behavior of LexA is unusual in that the steepness of this salt dependence (delta log K omega/delta log [NaCl]) is much more pronounced at slightly acidic pH values as compared to that at neutral or slightly alkaline pH. The behavior is not easily understood in terms of the current theories on the electrostatic contribution to protein-DNA interactions on the basis of polyelectrolyte theory. A comparison of the overall binding constant K omega of the entire LexA repressor and its DNA binding domain reveals that LexA binds only 20-50-fold stronger under a wide variety of salt and pH conditions. This result tends to demonstrate further that the additional energy due to the dimerization of LexA via the carboxy-terminal domain should be rather weak as expected from the small dimerization constant of LexA (2 X 10(-4) M-1).