The bipartite architecture of the sRNA in an archaeal box C/D complex is a primary determinant of specificity.
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.
Hardin
John W
JW
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
Batey
Robert T
RT
D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D020137:Base Pair Mismatch; D001483:Base Sequence; D024202:Electrophoretic Mobility Shift Assay; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D019642:RNA, Archaeal / Q000737:chemistry*; D020537:RNA, Small Nucleolar / Q000737:chemistry*; D020718:Ribonucleoproteins, Small Nucleolar / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D019843:Archaeal Proteins; D019642:RNA, Archaeal; D020537:RNA, Small Nucleolar; D020718:Ribonucleoproteins, Small Nucleolar; D012313:RNA; D008780:Methyltransferases; C052744:RNA 2'-O-methyltransferase