Recent approaches to probe functional groups in ribonuclease P RNA by modification interference |
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Authors: | Wolf-Dietrich Hard Jens M Warnecke Roland K Hartmann |
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Institution: | (1) School of Medicine, Dept. of Microbiology, SUNY at Stony Brook, 11794-5222 Stony Brook, NY, USA;(2) Institut für Biochemie, Medizinische Universität zu Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany |
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Abstract: | Modification interference is a powerful method to identify important functional groups in RNA molecules. We review here recent developments of techniques to screen for chemical modifications that interfere with (i) binding of(pre-)tRNA to bacterial RNase P RNA or (ii) pre-tRNA cleavage by this ribozyme. For example, two studies have analyzed positions at which a substitution of sulfur for thepro-Rp oxygen affects tRNA binding 1] or catalysis 2]. The results emphasize the functional key role of a central core element present in all known RNase P RNA subunits. The four sulfur substitutions identified in one study 2] to inhibit the catalytic step also interfered with binding of tRNA toE. coli RNase P RNA 1]. This suggests that losses in binding energy due to the modification at these positions affect the enzyme-substrate and the enzyme-transition state complex. In addition, the two studies have revealed, for the first time, sites of direct metal ion coordination in RNase P RNA. The potentials, limitations and interpretational ambiguities of modification interference experiments as well as factors influencing their outcome are discussed.Abbreviations nt
nucleotide(s)
- PAGE
polyacrylamide gel electrophoresis |
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Keywords: | modification interference Ribonuclease P RNA Rp-phosphorothioates |
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