title: Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.
abstract: RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product.
authors.affiliation: European Molecular Biology Laboratory, SCB Unit, D-69117 Heidelberg, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Graziadei
authors.affiliation: European Molecular Biology Laboratory, SCB Unit, D-69117 Heidelberg, Germany.
authors.forename: Pawel
authors.initials: P
authors.lastname: Masiewicz
authors.affiliation: European Molecular Biology Laboratory, SCB Unit, D-69117 Heidelberg, Germany.
authors.forename: Audrone
authors.initials: A
authors.lastname: Lapinaite
authors.affiliation: European Molecular Biology Laboratory, SCB Unit, D-69117 Heidelberg, Germany Leibniz University Hannover, Centre for Biomolecular Drug Research, D-30167 Hannover, Germany Helmholtz Centre for Infection Research, Group of Structural Chemistry, D-38124 Braunschweig, Germany.
authors.forename: Teresa
authors.initials: T
authors.lastname: Carlomagno
mesh_terms: D001105:Archaea / Q000201:enzymology*; D004798:Enzymes / Q000378:metabolism*; D008745:Methylation; D009154:Mutation; D009690:Nucleic Acid Conformation; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics*
chemical_list: D004798:Enzymes; D012335:RNA, Ribosomal
keywords: 2′-O methylation; RNA modification; box C/D RNP; regulatory mechanism