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Cottrell J.W.,Scripps Research Institute | Scott L.G.,Cassia, Llc | Fedor M.J.,Scripps Research Institute
Journal of Biological Chemistry | Year: 2011

Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis.Wedeveloped a way to determine microscopic pK a values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pK a for ionization of 8azaA at position 38 with the apparent pK a for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK a values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5′-oxygen-phosphorus bond. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Where chromatographic separation of sucrose from molasses produced is employed, the color of the molasses feed to the molasses separator operation has a relatively large effect on the color of the sucrose enriched extract produced from the separation process and, as a result, on the overall efficiency of sugar recovery from the molasses separation process. The subsequent sugar end processing efficiency of the separator sucrose enriched extract product to granulated sugar is quite dependent on the color loading of the extract being processed. This study evaluates the effect of manipulating and controlling the pH value of the A (high green) run-off syrup, B (intermediate) green run-off syrup and final molasses produced to achieve minimal color rise and a low final molasses color from the thick juice processed in the sugar end. The deliberate and precise control of green syrup pH value will produce molasses of the desired color relative to the quality of the beets being processed and the thick juice produced from such beet quality. It is further shown that the color of extract produced is highly dependent on the color of molasses feed to the molasses separation operation.

Viladoms J.,Scripps Research Institute | Scott L.G.,Cassia, Llc | Fedor M.J.,Scripps Research Institute
Journal of the American Chemical Society | Year: 2011

Active-site guanines that occupy similar positions have been proposed to serve as general base catalysts in hammerhead, hairpin, and glmS ribozymes, but no specific roles for these guanines have been demonstrated conclusively. Structural studies place G33(N1) of the glmS ribozyme of Bacillus anthracis within hydrogen-bonding distance of the 2′-OH nucleophile. Apparent pK a values determined from the pH dependence of cleavage kinetics for wild-type and mutant glmS ribozymes do not support a role for G33, or any other active-site guanine, in general base catalysis. Furthermore, discrepancies between apparent pK a values obtained from functional assays and microscopic pK a values obtained from pH-fluorescence profiles with ribozymes containing a fluorescent guanosine analogue, 8-azaguanosine, at position 33 suggest that the pH-dependent step in catalysis does not involve G33 deprotonation. These results point to an alternative model in which G33(N1) in its neutral, protonated form donates a hydrogen bond to stabilize the transition state. © 2011 American Chemical Society.

Scott L.G.,Cassia, Llc | Hennig M.,University of North Carolina at Chapel Hill
Methods in Enzymology | Year: 2016

Naturally occurring RNA lacks fluorine-19 (19F), thus, their specifically fluorinated counterparts are particularly well suited to noninvasively monitoring the dynamic conformational properties and ligand-binding interactions of the RNA. For nuclear magnetic resonance (NMR) spectroscopy, 19F-NMR of fluorine-substituted RNA provides an attractive, site-specific probe for structure determination in solution. Advantages of 19F include high NMR sensitivity (83% of 1H), high natural abundance (100%), and the extreme sensitivity of 19F to the chemical environment leading to a large range of chemical shifts. The preparation of base-substituted 2-fluoropurine and 5-fluoropyrimidine 5′-triphosphates (2F-ATP/5F-CTP/5F-UTP) can be carried out using efficient enzymatic synthesis methods. Both pyrimidine analogs, 5-fluorouridine and 5-fluorocytidine, as well as, 2-fluoroadenosine are readily incorporated into RNA transcribed in vitro using T7 RNA polymerase. © 2016 Elsevier Inc. All rights reserved.

Havelund J.F.,University of Southern Denmark | Havelund J.F.,Danish Technological Institute DTI | Giessing A.M.B.,University of Southern Denmark | Hansen T.,University of Southern Denmark | And 4 more authors.
Journal of Molecular Biology | Year: 2011

Complete characterization of a biomolecule's chemical structure is crucial in the full understanding of the relations between their structure and function. The dominating components in ribosomes are ribosomal RNAs (rRNAs), and the entire rRNA-but a single modified nucleoside at position 2501 in 23S rRNA-has previously been characterized in the bacterium Escherichia coli. Despite a first report nearly 20 years ago, the chemical nature of the modification at position 2501 has remained elusive, and attempts to isolate it have so far been unsuccessful. We unambiguously identify this last unknown modification as 5-hydroxycytidine-a novel modification in RNA. Identification of 5-hydroxycytidine was completed by liquid chromatography under nonoxidizing conditions using a graphitized carbon stationary phase in combination with ion trap tandem mass spectrometry and by comparing the fragmentation behavior of the natural nucleoside with that of a chemically synthesized ditto. Furthermore, we show that 5-hydroxycytidine is also present in the equivalent position of 23S rRNA from the bacterium Deinococcus radiodurans. Given the unstable nature of 5-hydroxycytidine, this modification might be found in other RNAs when applying the proper analytical conditions as described here. © 2011 Elsevier Ltd. All rights reserved.

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