The hairpin ribozyme is an example of a small catalytic RNA which catalyses the endonucleolytic transesterification of RNA in a highly sequence-specific manner. The hairpin ribozyme, in common with all other small ribozymes such as the hammerhead, requires the presence of a divalent metal ion co-factor (typically magnesium) for the reaction to take place. To investigate the role of magnesium ions in the hairpin catalysed reaction we have synthesised two epimeric modified substrates in which a phosphorothioate replaces the scissile phosphodiester bond. Previously, Burke and co-workers have reported that no thio-effect is observed with the Rp-phosphorothioate isomer. We observe the absence of a thio-effect with both diastereomeric phosphorothioate hairpin substrates. Furthermore we report that inert cobalt (III) complexes are capable of supporting the hairpin ribozyme reaction, with a similar efficiency to Mg2+,even in the presence of EDTA. Variation of the net charge on the inert cobalt complex does not change the observed rate of reaction. These results suggest that metal ions play a passive role in the hairpin ribozyme catalysed reaction and are probably required for structural purposes only. This places the hairpin ribozyme in a different mechanistic class to other small ribozymes such as the hammerhead.
The formation of hydrogenbonded complexes between three different compounds has been investigated by 1 H NMR spectroscopy. Titration experiments for binary and ternary mixtures show that these compounds form a termolecular complex in chloroform. The complexation-induced changes in chemical shift indicate that the structure of the ternary complex is similar to the hydrogen-bonded structures found in the simple binary mixtures. However, the association constant for the formation of the ternary complex is significantly larger than that expected based on the stabilities of the binary complexes: the association constant increases by a factor of three, equivalent to a stabilisation of 1 ± 2 kJ mol
À1. An explanation for this phenomenon is that the formation of a small hydrogen-bond network polarises the hydrogen-bonding groups and thereby increases the strengths of the individual hydrogenbonding interactions.
The measurement of internal gas temperature by the technique of acoustic pyrometry potentially offers excellent resolution due to its dependence on the measurement of time. The accuracy of the technique in combustion applications, however, must be questioned given its dependence on the acoustic constant, C a . In a typical combustion application, where the exact composition of the gas along the measuring path may not be known, this is of particular concern. Systematic errors arise due to the deviations of the actual gas properties from those assumed.Analysis of the variation of the acoustic constant for the products of combustion of typical fuels is reported as a function of temperature, mixture ratio and oxidant composition. From this it is possible to quantify the level of systematic error and hence the accuracy of the technique for a general case.A strong dependence of C a is observed with temperature but this may easily be taken into account in the measurement using correction factors. A far lesser dependence is observed on combustion mixture ratio, moisture and air humidity for a wide range of fuels and oxidant compositions. The systematic error resulting from ignoring these factors over an extensive range of values is better than ±2% for most applications.
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