Molecular modelling of the GIR1 branching ribozyme gives new insight into evolution of structurally related ribozymes

Twin-ribozyme introns contain a branching ribozyme (GIR1) followed by a homing endonuclease (HE) encoding sequence embedded in a peripheral domain of a group I splicing ribozyme (GIR2). GIR1 catalyses the formation of a lariat with 3 nt in the loop, which caps the HE mRNA. GIR1 is structurally related to group I ribozymes raising the question about how two closely related ribozymes can carry out very different reactions. Modelling of GIR1 based on new biochemical and mutational data shows an extended substrate domain containing a GoU pair distinct from the nucleophilic residue that dock onto a catalytic core showing a different topology from that of group I ribozymes. The differences include a core J8/7 region that has been reduced and is complemented by residues from the pre-lariat fold. These findings provide the basis for an evolutionary mechanism that accounts for the change from group I splicing ribozyme to the branching GIR1 architecture. Such an evolutionary mechanism can be applied to other large RNAs such as the ribonuclease P

How much hydration is necessary for the stabilisation of DNA-duplex?

A combination of NOESY and ROESY experiments show that the higher stabilities (T(m)) of phenazine tethered matched (2) and G-A mismatched (4) DNA duplexes are due to the decrease of the exchange-rate (i.e. increase of the life-time) of the imino-protons and the reduced water activity in their minor grooves compared to their non-tethered counterparts (1) and (3)

Wheat starch carbamate: Production, molecular characterization, and film forming properties

Wheat starch carbamates of different degrees of substitution were produced in laboratory experiments and for the first time their film forming performance were investigated. The carbamation reaction between urea and starch was investigated using a factorial design. Long reaction time, 2 h, and high urea content, 10 and 25%, resulted in a high degree of substitution, 0.07 and 0.15, respectively. These starch carbamates were assumed to be cross-linked and showed best film forming properties resulting in continuous and firm films. Furthermore, a high degree of carbamate substitution favored a decrease in glass transition temperature (T-g) in cast films. The addition of acid as a catalyst for carbamation of starch produced inconsistent results and mainly lead to degradation of starch molecules that caused brittle films. FTIR and C-13 NMR analyses confirmed the covalent bonding between urea and starch in starch carbamates. In a final step, production of starch carbamates was successfully scaled up. A potential industrial use of these starches is as oxygen barrier in multilayer food packaging

Distribution and metabolism in healthy volunteers of disodium azodisalicylate, a potential therapeutic agent for ulcerative colitis.

A series of experiments has been performed in healthy male volunteers to investigate the disposition of orally administered disodium azodisalicylate, a potentially useful drug for the treatment of ulcerative colitis. The drug was given by mouth in doses of up to 2 g a day for six weeks and there were no adverse effects. Serum concentrations of the intact compound were low and the serum half-time was 4-12.8 days, probably because of a combination of a low clearance rate and a high apparent volume of distribution. Less than 5% of the ingested dose was excreted unchanged in the urine. Circulating concentrations of 5-ASA and N-acetyl-5-ASA were low and 30% of the equivalent daily dose was excreted in the urine, predominantly as N-acetyl-5-ASA. In most subjects more than 30% of the equivalent daily dose of 5-ASA was recovered from the faeces, either as 5-ASA itself or as the acetylated derivative. As 5-ASA has been shown to be the active therapeutic moiety of sulphasalazine, disodium azodisalicylate appears to be suitable for therapeutic trial in ulcerative colitis

Solid-phase synthesis of a 6-phenylquinolin-2(1H)-one library directed toward nuclear hormone receptors

A library of 6-phenylquinolin-2(1H)-ones with diversity at position I and the ortho, meta, and para positions of the pendant phenyl ring has been synthesized using solid-phase parallel synthetic techniques. A key step in the synthesis of the library is a tandem alkylation cleavage in which diversity can be introduced at position 1 simultaneously to the cleavage from the resin. The yields of this step were significantly improved over what has previously been reported by addition of cesium carbonate to scavenge the acid that is formed during the reaction. Furthermore, we have shown that the solid support linkage is tolerant to Suzuki coupling and etherification reaction conditions and that selective cleavage of the linkage can take place in the presence of esters. The resulting 6-phenylquinolin-2(1H)-one library was screened against a panel of nuclear hormone receptors (androgen, estrogen alpha and beta isoforms, glucocorticoid, mineralocorticoid, and progesterone). Certain members of this library display moderate affinity for several of these receptors, and consequently, the 6-phenylquinolin-2(1H)-one core of the library may be considered a privileged structure for nuclear hormone receptors. In contrast, other members of the library display high selectivity for a particular receptor. The highest affinity ligand (9{2,1,1}) possesses an affinity of 330 nM for the androgen receptor, whereas the most selective ligand (9{2,4,1}) displays an affinity of 900 nM for the androgen receptor and a selectivity of 140-fold over the next highest affinity receptor

Crenarchaeal chromatin proteins Cren7 and Sul7 compact DNA by inducing rigid bends

Archaeal chromatin proteins share molecular and functional similarities with both bacterial and eukaryotic chromatin proteins. These proteins play an important role in functionally organizing the genomic DNA into a compact nucleoid. Cren7 and Sul7 are two crenarchaeal nucleoid-associated proteins, which are structurally homologous, but not conserved at the sequence level. Co-crystal structures have shown that these two proteins induce a sharp bend on binding to DNA. In this study, we have investigated the architectural properties of these proteins using atomic force microscopy, molecular dynamics simulations and magnetic tweezers. We demonstrate that Cren7 and Sul7 both compact DNA molecules to a similar extent. Using a theoretical model, we quantify the number of individual proteins bound to the DNA as a function of protein concentration and show that forces up to 3.5 pN do not affect this binding. Moreover, we investigate the flexibility of the bending angle induced by Cren7 and Sul7 and show that the protein-DNA complexes differ in flexibility from analogous bacterial and eukaryotic DNA-bending proteins.Publisher PDFPeer reviewe

The solution structure of a 3'-phenazinium (Pzn) tethered DNA-RNA duplex with a dangling adenosine: r(5'G-AUUGAA3'):d(5'TCAATC3'-Pzn).

The 3'-Pzn group tethered to an oligo-DNA stabilizes a DNA-RNA hybrid duplex structure by 13 degrees C compared to the natural counterpart. This report constitutes the first full study of the conformational features of a hybrid DNA-RNA duplex, which has been possible because of the unique stabilization of this rather small duplex by the tethered 3'-Pzn moiety (Tm approximately 40 degrees C from NMR). In this study, a total of 252 inter- and intra-strand torsional and distance constraints along with the full NOE relaxation matrix, taking into account the exchange process of imino and amino protons with water, have been used. The 3'-Pzn-promoted stabilization of the DNA-RNA hybrid duplex results in detailed local conformational characteristics such as the torsion angles of the backbone and sugar moieties that are close to the features of the other natural DNA-RNA hybrids (i.e. sugars of the RNA strand are 3'-endo, but the sugars of the DNA strand are intermediate between A- and B-forms of DNA, 72 degrees < P < 180 degrees; note however, that the sugars of our DNA strand have a C1-exo conformation: 131 degrees < P < 154 degrees). This study suggests that 3'-Pzn-tethered smaller oligo-DNA should serve the same purpose as a larger oligo-DNA as a antisense inhibitor of the viral mRNA. Additionally, these types of tethered oligos have been found to be relatively more resistant to the cellular nuclease. Moreover, they are taken up quite readily through the cellular membrane (14) compared to the natural counterparts