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S Brahms

Publications and source records attributed to S Brahms.

18 recordsLinked to original sources
PubMed1995

Binding of the transcription activator NRI (NTRC) to a supercoiled DNA segment imitates association with the natural enhancer: an electron microscopic investigation.

Electron microscopic visualization indicates that the transcription activator NRI (NTRC) binds with exceptional selectivity and efficiency to a sequence-induced superhelical (spiral) segment inserted upstream of the glnA promoter, accounting for its observed ability to substitute for the natural glnA enhancer. The cooperative binding of NRI to the spiral insert leads to protein oligomerization which, at higher concentration, promotes selective coating of the entire superhelical segment with protein. Localization of NRI at apical loops is observed with negatively supercoiled plasmid DNA. With a linear plasmid, bending of DNA is observed. We confirm that NRI is a DNA-bending protein, consistent with its high affinity for spiral DNA. These results prove that spiral DNA without any homology to the NRI-binding sequence site can substitute for the glnA enhancer by promoting cooperative activator binding to DNA and facilitating protein oligomerization. Similar mechanisms might apply to other prokaryotic and eukaryotic activator proteins that share the ability to bend DNA and act efficiently as multimers.

Bacterial Proteins↗
PubMed1995

A sequence-induced superhelical DNA segment serves as transcriptional enhancer.

The initiation of transcription at the sigma 54-dependent promoter glnAp2 of Escherichia coli is activated by the protein NR1(NTRC)-phosphate, which binds to two sites located upstream of the promoter that together constitute an enhancer. The cooperative binding facilitates the oligomerization of NR1-phosphate endowing it with the ATPase activity required for its ability to serve as transcriptional activator. We show here that these sites can be replaced by sequence-dependent superhelical inserts, lacking any homology to the nucleotide sequence of the enhancers. These superhelical inserts, irrespective of their chirality, are as effective as the paired sites in binding NR1-phosphate and in stimulating its oligomerization. We conclude that a specific sequence of nucleotides and the three-dimensional structure of DNA can determine its affinity for the NR1 activator protein capable of binding to DNA.

Adenosine Triphosphatases↗
PubMed1992

Investigations on the dynamic structures of adenine- and thymine-containing DNA.

The structures of poly(dA-dT), poly(dA-dBr5U) and of poly(dA).poly(dT) have been investigated in solution and in fibers, by Raman spectroscopy. Both the alternating poly(dA-dT), poly(dA-dBr5U) and non-alternating poly(dA).poly(dT) exhibit, in the region of sugar phosphate backbone vibrations, two bands of almost equal intensity at about 841 cm-1 and 817 cm-1. The analysis of the characteristic bands of thymine residues that are sensitive to sugar puckers gives indication of a significant displacement from the C(2')-endo conformer suggesting the adoption of alternative conformers such as O(4')-endo. In contrast, the diagnostic Raman bands for the sugar pucker of adenine residues suggest, instead, predominant adoption of C(2')-endo conformations. These Raman results are compatible with rapid dynamic changes of sugar puckers between C(2')-endo and O(4')-endo for the thymidine (and uridine) residues, whereas in adenine residues the sugar puckers fluctuate around the C(2')-endo pucker in all synthetic DNA molecules studied. Molecular dynamics simulations, performed on six different starting models using two distance-dependent dielectric functions epsilon(r) = 4 r and a sigmoidal dependence), all gave similar dynamic behavior in agreement with these Raman data and their interpretation. The mean calculated pseudorotation phases of the adenine residues are systematically higher (around C(2')-endo) than those of the thymine residues (close to O(4')-endo-C(1')-exo). Besides, the mean lifetimes of the thymine residues are 1.5 to 2.0-fold higher in the O(4')-endo than in the C(2')-endo domain, while those of the adenine residues are two to threefold higher in the C(2')-endo than in the O(4')-endo domain. In the Raman spectra of the alternating poly(dA-dBr5U), the splitting of a band into two components arising from the two contributions of ApBr5U and Br5UpA provides strong evidence for a repeating dinucleotide structure in solution. The calculated twist values averaged over the simulation runs are also systematically higher in the 5'T-A3' step (39 degrees) than in the 5'A-T3' step (33 degrees). Simultaneously, the calculated roll values are positive in the 5'T-A3' step (6 degrees) and negative in the 5'A-T3' step (-9 degrees), while the propeller twist values are about the same (-11 degrees to -16 degrees). On the other hand, in the homopolymer, the average twist value is close to 36 degrees with the roll angle close to 0 degrees and large propeller twist values (-20 degrees).

Adenine↗
PubMed1990

DNA with adenine tracts contains poly(dA).poly(dT) conformational features in solution.

The conformation of DNA's with adenine-thymine tracts exhibiting retardation in electrophoretic migration and considered as curved were investigated in solution by CD and RAMAN spectroscopy. The following curved multimers with adenine tracts but of different flanking sequences d(CA5TGCC)n, d(TCTCTA6TATATA5)n, d(GA4T4C)n yield CD spectroscopic features indicating a non-B structure of the dA.dT tract with similarities to polyd(A).polyd(T). We suggest that adenine-thymine bases in these multimers contain some of the distinctive conformational features of poly(A).polyd(T) probably with large propeller twist found by NMR (Behling and Kearns, 1987) and by X-ray diffraction on oligonucleotides containing a tract of adenines (Nelson et al. 1987, Coll et al; 1987; DiGabriele et al. 1989). Some elements of distinctive CD features of the contiguous adenines run are also observed in the straight multi-9-mer d(CA5GCC)n which lacks in-phase relation to the helical repeat. Despite the presence of the TpA step in the straight multimer d(GT4A4)n, the altered dA.dT conformation is not completely destroyed. Interruption of adenine tract by a guanine in d(CAAGAATGCC)n leads to a B-like conformation and to a normal electrophoretic mobility. The Raman spectra reveal a rearrangement of the sugar-phosphate backbone of dA.dT tract in the multimer d(CA5TGCC)n with respect to that of polydA.polydT. This is reflected in the presence of an unique Raman band associated to C2'-endo sugar with a predominant contribution of C1'-exo puckering which is exhibited by the multimer whereas two distinct Raman bands characterize poly(dA).poly(dT) backbone conformation.

Adenine↗
PubMed1989

Structural changes in positively and negatively supercoiled DNA.

The effect of superhelical constraint on the structure of covalently closed circular DNA (cccDNA; pBR322) with positive and negative writhe (superturn) has been investigated as a function of decreasing and increasing specific linking difference (mean superhelical density sigma). At low and moderate negative superhelical densities sigma, the overall average structure is maintained in an unwound B-form slightly modified. The overwound cccDNAs with positive writhe differ from those with negative writhe by an absence of cruciform structure. At high negative densities of supercoiling different changes involving the reversal of twist handedness are shown to lead to the formation of DNA segments in a conformation identical to the left-handed component of form V DNA.

Circular Dichroism↗
PubMed1989

The synthetic DNA duplex of poly d(Abr5U).poly d(Abr5U) adopts an A-DNA-like structure.

An X-ray fiber diffraction study of the synthetic DNA duplex poly d(Abr5U).poly d(Abr5U) shows that its sodium salt adopts an unexceptional A-DNA-like structure. Similar to A-DNA, two molecules are packed in a monoclinic unit cell (a = 2.23 nm, b = 4.14 nm, c = 5.61 nm and alpha = beta = gamma = 90 degrees) of space group C2. Because of its dinucleotide chemical motif, the c-repeat is twice that in A-DNA but, notably, corresponding backbone conformation angles of adjacent nucleotides are almost identical. This is in marked contrast to many B-like conformations of polydinucleotides.

Crystallization↗
PubMed1985

Activation and inhibition of transcription by supercoiling.

Stimulation of transcriptional activity in vitro is observed at low and moderate negative superhelical densities up to the level of the natural superhelical form of the plasmid pBR322. We have isolated and identified three specific transcription products: ampicillinR RNA, tetracyclineR RNA and "Rep" RNA. Their enhancement of transcription occurs at different levels of superhelicity, suggesting a sequence-dependent structural alteration of promoters upon changes of axial writhe, which may generate kink formation. The activation of transcription is drastically inhibited at higher specific linking differences exceeding that of the natural superhelical form of pBR322, which is correlated with a transition from the right-handed B to a left-handed DNA form of particular sequences induced by supercoiling. We have identified a new stop point in the beta-lactamase coding sequence composed of eight alternating purine-pyrimidine residues which, at higher torsional stress, causes transcription to stop, leading to the synthesis of a short RNA of about 55 nucleotides instead of AmpR RNA (about 580 nucleotides). In the "Rep" promoter, two alternating purine-pyrimidine segments are found, which conformational change at higher superhelical densities may be implicated in repression of "Rep" RNA synthesis. The enhancement and inhibition of transcription by supercoiling support the role of energetic and structural changes in topologically constrained DNA as elements of a control mechanism.

Autoradiography↗
PubMed1983

Torsional stress induces left-handed helical stretches in DNA of natural base sequence: circular dichroism and antibody binding.

Above a threshold of torsional stress, the c.d. spectrum of covalently closed circular DNA of natural base sequence acquires a Z-like contribution and antibodies raised against Z-DNA are bound. Mapping of the antibody binding sites by electron microscopy reveals sites which correlate with stretches enriched in alternating purine-pyrimidine sequences and GC base pairs.

Animals↗
PubMed1981

Conformation of DNA in chromatin reconstituted from poly [d(A-T)] and the core histones.

Present results provide direct evidence of the nature of a conformational change in DNA when nucleosomes are formed from core histones and poly [d(A-T)]. First, we have found some features which have characteristic aspects of the A like conformation of DNA. Thus, an increased contribution due to a sugar conformation close to C3'-endo puckering is detected in the Raman spectra. In addition, the circular dichroism (C.D.) spectra of reconstituted chromatin with poly [d(A-T)] exhibits an increases intensity at about 262 nm. A second feature acquired by poly [d(A-T)] in nucleosome formation from core histones is related to the presence of a negative band at about 280 nm in the C.D.spectra. The nature of this change is correlated with a DNA conformation characterized by a decreased number of base pairs per turn (28,29). This indicates that these two features of reconstituted nucleosomes reflect the presence of two types of DNA conformations, which overall form is of the B type (22,36).

Animals↗
PubMed1979

Effect of 5-alkyl substitution of uracil on the thermal stability of poly [d(A-r5U)] copolymers.

The thermal transition of poly[d(A-r5U)] polydeoxynucleotides (where r was a hydrogen atom, or a methyl, ethyl, n-propyl, n-butyl or n-pentyl group) was studied by measuring the derivative melting profiles of the polymers in the range of 0.01--0.36 M K+, at pH 6.8. According to the Tm values, polydeoxynucleotide analogues show lower thermal stability than poly[d(A-T)] at any counterion concentration applied. At a given salt concentration, Tm of the alkyl analogues decreased as the number of carbon atoms (n) in the r substituent of poly[d(A-r5U)] increased. 1/Tm plotted against against 1/n yielded a linear relationship. Cooperativity of the melting of all poly[d(A--U)] analogues decreased with the increase of salt concentration in the solution. This change depended again on 5-substitution of the uracil moiety of poly[d(A-U)]. Smallest decrease was observed in the case of poly[d(A--U)] whereas largest decrease was shown by poly[d(A-pe5U)] (pe=pentyl group).

Alkylation↗
PubMed1977

Identification of beta,beta-turns and unordered conformations in polypeptide chains by vacuum ultraviolet circular dichroism.

Different conformations of polypeptides were characterized by measurements of the circular dichroism (CD) extended into the vacuum ultraviolet region. (i) The linear beta-pleated sheet structure was characterized in a broad ultraviolet region down to 165 nm by examination of copolypeptides composed of alternating hydrophobic and hydrophilic amino-acid residues, e.g., poly(Lys-Leu-Lys-Leu). A short-wavelength intense band was found at about 169 nm, which is characteristic of beta-pleated sheet conformation. (ii) The beta-turns were experimentally measured using poly(Ala(2)-Gly(2)) in a broad spectral region down to 165 nm with accuracy. The observed CD spectrum is in excellent qualitative agreement with the theoretical curve calculated by Woody for the beta-turns of type II and/or I of Venkatachalam. The similarity in shape between the theoretical curve and the observed CD spectra suggests a dominance of beta-turn segments in the poly(Ala(2)-Gly(2)) structure. The presence of beta-turns in poly(Ala(2)-Gly(2)) is also in agreement with the characterization of this polypeptide by solid state methods (electron microscopy and x-ray diffraction). The CD spectrum of beta-turns is characterized by a very intense band at 207.5 nm and strong negative bands at 191 and 169 nm. Copolypeptides such as poly(Ala(2)-Gly(3)) and poly(Ala(3)-Gly(3)) yielded a similar type of CD spectrum, analysis of which indicates that a large fraction of their residues is contained in beta-turn regions. (iii) The CD spectrum of the unordered chain of these alternating copolypeptides in salt-free solution is observed in the vacuum ultraviolet region.

Circular Dichroism↗
PubMed1977

Effect of non-histone proteins on thermal transition of chromatin and of DNA.

The effect of chromatin non-histone protein on DNA and chromatin stability is investigated by differential thermal denaturation method. 1) Chromatin (rat liver) yields a multiphasic melting profile. The major part of the melting curve of this chromatin is situated at temperatures higher than pure DNA, with a distinct contribution due to nucleosomes melting. A minor part melts at temperatures lower than DNA which may be assigned to chromatin non-histone protein-DNA complex which destabilized DNA structure. 2) Heparin which extracts histones lowers the melting profile of chromatin and one observes also a contribution with a Tm lower that of pure DNA. In contrast, extraction on non-histone proteins by urea supresses the low Tm peak. 3) Reconstitution of chromatin non-histone protein-DNA complexes confirms the existence of a fraction of chromatin non-histone protein which lowers the melting temperature when compared to pure DNA. It is concluded that chromatin non-histone proteins contain different fractions of proteins which are causing stabilizing and destabilizing effect on DNA structure.

Animals↗
PubMed1976

Nature of conformational changes in poly[d(A-T)-d(A-T)] in the premelting region.

The conformation of the synthetic DNA, poly-[d(A-T)-d(A-T)], has been investigated both in the solid state and in dilute aqueous solutions at different temperatures below its melting point. The change of the circular dichroism (CD) spectra of poly[d(A-T)-d(A-T)] solutions with decreasing temperatures from just below the melting point to 0 degrees involves a specific decrease of the intensity of the 262 nm CD band. This conformational change has been assigned to a gradual and partial transition from the B to C form, on the basis of the following results: (i) By the use of infrared dichroism measurements on oriented films we have defined humidity and salt conditions under which B and C forms of poly[d(A-T)-d(A-T](Li+) are stable. In addition, we find that ammonium salts induce the C form of poly[d(A-T)-d(A-T)] even at high relative humidity. (ii) CD studies of the films of the lithium salt of poly[d(A-T)-d(A-T)] under the same conditions have given CD spectra corresponding to the B and C forms of this polynucleotide. In addition, the CD spectrum of the ammonium salt of poly[d(A-T)-d(A-T)] in solution approaches that of the C form in films. (iii) The conformational change of poly[d(A-T)-d(A-T)] as a function of temperature can be entirely explained on the basis of changes in the double-stranded base-paired structure. Our data rule out hydrogen bond breaking and unstacking or "breathing" as an explanation of the premelting changes. Curves of the continuous variation of CD(epsilon at 262 nm) as a function of temperature (from 0 degrees to the melting zone) show similar slopes in the presence of different agents stabilizing the double-stranded structure, such as Mg++, or at different salt concentration (KCl), indicating that the nature of the process is independent of ionic strength. Some specific effects were observed in the influence of certain neutral salts; ammonium induces the C form whereas magnesium favors the B form. CD data give direct evidence that a DNA like poly[d(A-T)-d(A-T)] need not change conformation upon transition from a dilute aqueous solution to a highly hydrated (film/gel) solid state. The change of conformation begins only at a defined partial dehydration.

Circular Dichroism↗