Nucleic Acids Research Advance Access originally published online on September 19, 2006
Nucleic Acids Research 2006 34(18):5039-5051; doi:10.1093/nar/gkl644
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Nucleic Acids Research, 2006, Vol. 34, No. 18 5039-5051
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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The bipartite architecture of the sRNA in an archaeal box C/D complex is a primary determinant of specificity
Department of Chemistry and Biochemistry, University of Colorado Boulder, CO 80309, USA
*To whom correspondence should be addressed. Tel: +1 303 735 2159; Fax: +1 303 735 1347; Email: robert.batey{at}colorado.edu
Received July 28, 2006. Accepted August 21, 2006.
The archaeal box C/D sRNP, the enzyme responsible for 2'-O-methylation of rRNA and tRNA, possesses a nearly perfect axis of symmetry and bipartite structure. This RNP contains two platforms for the assembly of protein factors, the C/D and C'/D' motifs, acting in conjunction with two guide sequences to direct methylation of a specific 2'-hydroxyl group in a target RNA. While this suggests that a functional asymmetric single-site complex complete with guide sequence and a single box C/D motif should be possible, previous work has demonstrated such constructs are not viable. To understand the basis for a bipartite RNP, we have designed and assayed the activity and specificity of a series of synthetic RNPs that represent a systematic reduction of the wild-type RNP to a fully single-site enzyme. This reduced RNP is active and exhibits all of the characteristics of wild-type box C/D RNPs except it is nonspecific with respect to the site of 2'-O-methylation. Our results demonstrate that protein–protein crosstalk through Nop5p dimerization is not required, but that architecture plays a crucial role in directing methylation activity with both C/D and C'/D' motifs being required for specificity.
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