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Deuterium nuclear magnetic resonance studies of crambin crystals grown from deuterated solvent (2H2O/CH3CH2O2H or H2O/C2H3CH2OH) are reported. The extent to which the hydrate and exchangeable hydrogen atoms are dynamically disordered are then determined from the size of the residual deuterium quadrupole couplings, qcc. Rapid molecular reorientation (tau c-1 greater than 10(5) s-1) reduces the magnitude of the quadrupole coupling from its static value (216 kHz for solid water). We find that the room temperature spectrum of crambin is dominated by two features: a sharp line with very small residual quadrupolar coupling less than 3 kHz, and a broad pattern with a quadrupolar coupling in the range 185 to 195 kHz. The former is indicative of very nearly isotropically reorienting deuterons, whereas the latter is somewhat narrower than that observed for the amide deuterons of poly-gamma-benzyl-L-glutamate and thus indicative of deuterons that are almost but not completely stationary. By considering the nuclear magnetic resonance spectrum intensities along with the amino acid sequence, X-ray structure and the manner in which quadrupole couplings are reduced by dynamics, we conclude that the nuclear magnetic resonance signal from most of the water molecules of hydration are contained in the sharp line, i.e. reorient nearly isotropically in the crystalline protein. Unlike bulk water, which freezes abruptly in the manner of a phase transition, the water of hydration in crambin has a broad freezing range from 180 to 250K, as evidenced by the decreasing intensity of the sharp line that disappears at 180K. At temperatures between 150 and 200K, a typical hydrate molecule reorients at a rate comparable to the quadrupole coupling, 10(4) s-1 to 10(5) s-1, a process that occurs in hexagonal ice in the range of 240 to 270K. At 140K, the hydrate is stationary, tau c-1 less than 10(3) s-1. Studies of the protein crystallized from solvent deuterated only at the non-exchangeable methyl group of ethanol confirm that ethanol is in the lattice and show that this solvate behaves in much the same way as the hydrate. The refined X-ray structure has identified four ethanol solvate molecules. The deuterium spectrum at room temperature has a well-defined residual pattern with qcc = 2.2 kHz, i.e. a small-order parameter consistent with nearly isotropically reorienting molecules. The spectrum width broadens substantially only at temperatures below 200K and achieves the characteristic spectrum of a rotating methyl group with stationary C-C axis at 140K.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1989 Aug 20
PMID:Orientational ordering and dynamics of the hydrate and exchangeable hydrogen atoms in crystalline crambin. 281 Mar 59

Using proton relaxation and magnetization transfer from water we have measured the imino proton exchange kinetics in two dodecadeoxynucleotide duplexes. One is formed by the self-complementary sequence 5'-d(C-C-T-T-T-C-G-A-A-A-G-G), the other by the inverse sequence. The imino proton exchange rates are found to depend on the concentration of ammonia or imidazole, acting as basic catalysts of proton exchange. Extrapolation of exchange times to infinite catalyst concentration yields the base-pair lifetimes, for instance 40 milliseconds for the central G.C base-pair of the 5'-d(C-C-T-T-T-C-G-A-A-A-G-G) duplex and four milliseconds for its A.T neighbour, at 15 degrees C. These results differ markedly from those reported by other laboratories for similar deoxy compounds. An explanation of the discrepancy has been proposed recently. Differences between base-pair lifetimes indicate that opening is not co-operative. From the catalyst efficiency relative to exchange from isolated nucleosides, we estimate the dissociation constant of each base-pair, e.g. 0.3 x 10(-6) and 1.5 x 10(-5) at 15 degrees C, for the same G.C and A.T base-pairs. The lifetime and dissociation constant of corresponding base-pairs of the two duplexes are similar, except for the central G.C base-pair. This correlates with differences in the solution structures reported by others. We have completed the assignments of the imino protons and of the six cytosine amino protons of the 5'-d(G-G-A-A-A-G-C-T-T-T-C-C) 12-mer. A new base-pair numbering scheme is proposed.
J Mol Biol 1987 Aug 05
PMID:Proton exchange and base-pair lifetimes in a deoxy-duplex containing a purine-pyrimidine step and in the duplex of inverse sequence. 282 87

Structural features of pyrimidine.pyrimidine mismatches in the interior of oligonucleotide duplexes have been investigated by high resolution two-dimensional proton nuclear magnetic resonance (n.m.r.) spectroscopy. These studies were conducted on the self-complementary d(C-G-C-T-A-G-C-T-T-G-C-G) duplex (designated T.T 12-mer) and the self-complementary d(C-G-C-C-A-G-C-T-C-G-C-G) duplex (designated C.C 12-mer) containing T.T and C.C pairs located at identical positions four base-pairs from either end of the duplex. Proton n.m.r. studies on the T.T 12-mer duplex were undertaken in the neutral pH range, while studies on the C.C 12-mer duplex were recorded at acidic pH. The proton spectra narrowed considerably on lowering the pH below neutrality for the C.C 12-mer duplex. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) data sets have been recorded on the T.T 12-mer and C.C 12-mer duplexes in high salt H2O and D2O solution. The magnitude of the NOE crosspeaks and the directionality of the NOE connectivities demonstrate that both duplexes are right-handed with all bases, including those at the mismatch site, adopting an anti configuration about the glycosidic bond. The observed base and sugar proton chemical shifts suggest structural similarities for the trinucleotide segments centered about the T.T and C.C mismatches. A NOE is detected between the resolved imino protons of T4 and T9 at the mismatch site, consistent with formation of a stacked "wobble" T4(anti).T9(anti) pair in the T.T 12-mer duplex. A comparison of the imino proton chemical shift and NOE data suggests that the imino-carbonyl hydrogen bonds in the wobble T.T mismatch are weaker than the corresponding imino-carbonyl hydrogen bonds in the wobble G.T mismatch. The 4-amino protons of C4 and C9 at the mismatch site in the C.C 12-mer duplex do not exhibit the pattern of hydrogen-bonded and exposed protons separated by approximately 1.5 parts per million characteristic of cytidine amino protons involved in Watson-Crick G.C pairing. The experimental data are insufficient to differentiate between wobble C(anti).C+(anti) and other pairing possibilities for the mismatch in the C.C 12-mer duplex at acidic pH.
J Mol Biol 1988 Jul 05
PMID:Pyrimidine.pyrimidine base-pair mismatches in DNA. A nuclear magnetic resonance study of T.T pairing at neutral pH and C.C pairing at acidic pH in dodecanucleotide duplexes. 284 94

The homeo box gene even-skipped (eve) encodes a 376-amino-acid protein that binds with high affinity to sequences located near the 5' termini of the eve and en genes. The 5' en sites are A + T rich and contain copies of the 10-base-pair (bp) consensus sequence T-C-A-A-T-T-A-A-A-T. In contrast, the 5' eve sites are G + C rich and contain the 9-bp sequence T-C-A-G-C-A-C-C-G. Among the five different homeo box proteins that have been tested for binding, eve is unique in that it shows virtually equal preference for the A + T-rich 5' en binding sites and the G + C-rich 5' eve sites. Most of the other proteins bind with a relatively higher affinity to the en sites than to the eve sites. In an effort to identify the regions of the eve protein that are responsible for its efficient binding to both classes of recognition sequences, we analyzed the DNA-binding properties of various mutant eve proteins. These studies suggest that the homeo domain of the eve protein is responsible for both binding activities. However, mutations in distant regions of the protein influenced the binding behavior of the eve homeo domain and caused a reduction in binding to the G + C class of recognition sites. We propose that the protein context of the homeo domain can influence its DNA-binding properties.
Mol Cell Biol 1988 Nov
PMID:DNA-binding activities of the Drosophila melanogaster even-skipped protein are mediated by its homeo domain and influenced by protein context. 290 20

The human ribosomal non-transcribed spacers are 30 X 10(3) base-pairs (or 30 kb) in length with a limited length heterogeneity localized in a specific region downstream from the 3' end of the transcribed region. Total DNA digested with EcoRI and BamHI and hybridized with a probe containing the 3' end of the 28 S ribosomal RNA coding region shows four major bands of 3.9 kb, 4.6 kb, 5.4 kb and 6.2 kb. The 5.4 kb band is the most abundant in every individual, followed by the 4.6 kb band. The longest and the shortest size classes are less well-represented and may even be absent. Every individual shows his own pattern of relative abundance of non-transcribed spacer length classes that can be followed through generations. We decided to investigate the molecular structure of the heterogeneity region, in order to cast light onto the mechanisms underlying the origin and maintenance of this length heterogeneity. Pertinent spacer regions of eight ribosomal clones from two human genomic libraries were subcloned and analyzed by restriction mapping and nucleotide sequencing. In the minimal length class, there is a sequence of 700 base-pairs that appears to be tandemly duplicated once, twice or three times in the other length classes. This repeated DNA module contains a region consisting of repetitions of simple pyrimidine groups like C-T, C-T-T-T or C-C-C-T. DNA module repeats may differ by the length of this pyrimidine-rich region. However, these length variations are not continuous, as revealed by Southern transfer analysis of several individuals and different cloned gene units: instead, the repeated modules fall into two discrete length classes of about 700 base-pairs and 800 base-pairs. An imperfect duplication of a short sequence of 86/89 base-pairs is present at the boundary between the heterogeneity region and the upstream flanking region, representing a very ancient duplication event.
J Mol Biol 1985 May 25
PMID:Molecular analysis of the heterogeneity region of the human ribosomal spacer. 298 41

The globin gene family of Xenopus laevis comprises pairs of closely related genes that are arranged in two clusters, each pair of genes being co-ordinately and stage-specifically expressed. To get information on putative regulatory elements, we compared the DNA sequences and the chromatin conformation 5' to the co-ordinately expressed adult alpha-globin genes. Sequence analysis revealed a relatively conserved region from the cap site up to position -289, and further upstream seven distinct boxes of homology, separated by more diverged sequences or deletions/insertions. The homology boxes comprise 22 to 194 base-pairs showing 78 to 95% homology. Analysis of chromatin conformation showed that DNase I preferentially cuts the upstream region of both genes at similar positions, 5' to the T-A-T-A and the C-C-A-A-T boxes, only in chromatin of adult erythroblasts and erythrocytes, where adult globin genes are expressed, but not in chromatin of adult liver cells or larval erythrocytes, where these genes are silent. This suggests that cell- and stage-specific activation of these genes coincides with specific changes in chromatin conformation within the proximal upstream region. No difference was found in the nucleotide sequence within the DNase I hypersensitive region proximal to the adult alpha 1-globin gene in DNA from embryonic cells, in which this gene is inactive, and adult erythrocytes, expressing this gene.
J Mol Biol 1986 Mar 20
PMID:Conserved sequences and cell-specific DNase I hypersensitive sites upstream from the co-ordinately expressed alpha I- and alpha II-globin genes of Xenopus laevis. 301 54

The structure of the human gene encoding the aldolase B isozyme has been determined, including the sequence of 14,887 base-pairs. The 5'- and 3'-ends have been determined by S1 mapping. There is a single gene for this enzyme in humans that was determined from the sequence and restriction enzyme digestions of genomic DNA. The gene is 14,500 base-pairs long containing nine exons. In addition, 924 and 208 base-pairs of the 5'- and 3'-flanking region, respectively, have been determined. There is a high degree of conservation of nucleic acid sequence between aldolase B genes of human, rat and chicken. The conservation extends to untranslated and flanking regions, and includes the derived protein structures. In the 5'-flanking region there are several sequence elements that are conserved in vertebrate aldolase B genes in addition to the T-A-T-A and C-C-A-A-T boxes. These sequences may be involved in the co-ordinate and tissue-specific control of expression of this gene. Several possible polymorphic sites were detected in the sequence of the human gene which may be useful for linkage mapping of the human genome and diagnostic analysis of alleles in families with hereditary fructose intolerance.
Mol Biol Med 1986 Jun
PMID:Characterization of the human aldolase B gene. 301 56

The effect of hairpin (cruciform) size on the regulation of gene expression was investigated by cloning a series of palindromic sequences into the non-essential J-F intercistronic region of the bacteriophage phi X174 ins6 genome. Genetic stability of the insert sequence and its effect on the growth efficiency of the phage was used as an initial measure of the biological consequence of hairpin insertions. Multimers of increasing size of the BamHI linker sequence C-C-G-G-A-T-C-C-G-G were inserted into the PvuII site of the parental strain ins6. The largest hairpin that could be constructed and maintained in the phi X174 genome had a stem length of 22 base-pairs and a loop size of four nucleotides (linker tetramer). However, this structure proved to be disadvantageous to the phage and was rapidly deleted from its genome. Trimer inserts were more stable, but were eventually deleted also. Monomer and dimer inserts, though genetically stable, decreased the growth efficiency of the phage as judged by competitive growth experiments and measurements of burst size. The physical formation of these hairpins was shown by restriction digests of single-stranded DNA with BamHI and HpaII. We argue that these secondary structures form in vivo, at least in the single-stranded genome and the polycistronic mRNAs, and were responsible for the observed growth defects.
J Mol Biol 1986 May 20
PMID:Insertions of palindromic DNA sequences into the J-F intercistronic region of bacteriophage phi X174 interfere with normal phage growth. 301 60

We have analyzed the chromatin structure of a region that encompasses 14.4 X 10(3) base-pairs of the chicken histone H5 locus in adult erythroid cells at different stages of maturation. Seven of eight major lineage-specific DNase I-hypersensitive sites, some of which show complex substructure, were found in the flanking regions of the gene. The hypersensitivity of some of these sites is modulated during erythrocyte maturation in a way that parallels the transcriptional activity of the gene. DNase I, micrococcal nuclease, and S1 nuclease recognize the same regions, which differ from those cleaved by S1 on supercoiled plasmid DNA. This suggests that hypersensitivity of DNA in chromatin reflects a greater accessibility of the DNA rather than its altered conformation. The DNA sequence of some of the DNase I target sites contains repeated motifs, (T-C-C-C)2, (T-C-C)2, (T-G-G-G-G)2, which are found in the hypersensitive sites of other genes. Detailed analysis across sections of the H5 gene and flanking sequences revealed differences in the DNase I sensitivity of the different regions examined. Notably, the first one-third of the gene is more sensitive than the rest. The sequences downstream from the region where most RNA polymerases terminate transcription were found to be the most resistant.
J Mol Biol 1986 May 05
PMID:Fine analysis of the active H5 gene chromatin of chicken erythroid cells at different stages of differentiation. 302 21

One- and two-dimensional NMR studies at 300 MHz and 500 MHz were carried out on the two oligonucleotides d(C-C-G-A-A-T-T-C-G-G) and d(C-C-G-A-m6A-T-T-C-G-G) in aqueous solution. NMR spectra were observed at 10 mM sample concentration over the temperature range 273-368 K. Assignments are given of the base, H1', H2', H2", H3' and of some H4' resonances, based upon a combination of two-dimensional correlation spectra (COSY) and two-dimensional nuclear Overhauser effect spectra (NOESY); imino-proton resonances were assigned with the aid of a two-dimensional NOE experiment. Chemical shift vs temperature profiles were constructed in order to gain insight into the influence of N6-methylation of residue A(5) on the temperature-dependent conformational behaviour of the decamer and to determine thermodynamic parameters for the duplex-to-coil transition. The NOESY spectra, the imino-proton spectra and the shift profiles of the two compounds, under conditions where each forms a B-DNA-type duplex, are very similar. This is taken to indicate that the influence of N6-methylation of residue A(5) on the local structure of the duplex must be small. However, the temperature dependence of the (non-)exchangeable proton resonances of the two compounds reveals that methylation slows down the duplex-single-strand exchange. Furthermore, a thermodynamic analysis of the two compounds indicates that N6-methylation slightly decreases the stability of the duplex relative to the monomeric forms (Tm is reduced from 332 K down to 325 K at 10 mM sample concentration). Proton-proton couplings were obtained by means of one-dimensional and two-dimensional NMR experiments and were used in a conformational analysis of the sugar ring of each residue of the two compounds in the duplex form. The analysis indicated that all sugar rings display conformational flexibility in the intact duplex: population S-type sugar conformation ranges from 70% to 100%. A more refined analysis of the sugar rings of the parent compound revealed a sequence-dependent variation of the sugar geometry. This variation does not follow well the trend predicted by the Calladine/Dickerson sigma 3-sum rule [Dickerson, R. E. (1983) J. Mol. Biol. 166, 419-441; Calladine, C. R. (1982) J. Mol. Biol. 161, 343-352]; moreover the actual variations appear to be smaller in solution than those expected on the basis of known X-ray structures.
 ...
PMID:Influence of N6-methylation of residue A(5) on the conformational behaviour of d(C-C-G-A-A-T-T-C-G-G) in solution studied by 1H-NMR spectroscopy. 1. The duplex form. 302


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