© 2004 Heron Publishing—Victoria, Canada
In vitro selection of an archaeal RNase P RNA mimics natural variation
Daniel Williams (1, 2) and James W. Brown (1, 3)
1. Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, USA / 2. V.A. Medical Center, Emory University Medical Research, Rm 5A188, 1670 Clarimont Rd., Decatur, GA 30033, USA / 3. Author to whom correspondence should be addressed ([email protected]) / Received September 17, 2003; accepted October 15, 2003; published online November 3, 2003
Archaeal and bacterial RNase P RNAs are similar in sequence and secondary structure, but in the absence of protein, the archaeal RNAs are much less active and require extreme ionic conditions for activity. To assess how readily the activity of the archaeal RNA alone could be improved by small changes in sequence, in vitro selection was used to generate variants of a Methanobacterium formicicum RNase P RNA: Bacillus subtilus pre-tRNAAsp self-cleaving conjugate RNA. Functional variants were generated with a spectrum of mutations that were predominately consistent with natural variation in this RNA. Variants generated from the selection had cleavage rates comparable to that of wild type; variants with improved cleavage rates or lower ionic requirements were not obtained. This suggests that the RNase P RNAs of Bacteria and Archaea are globally optimized and the basis for the large biochemical differences between these two types of RNase P RNA is distributed in the molecule.
Keywords: Archaea, archaebacteria, ribonuclease P, ribozyme, SELEX.
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