Abstract
In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are responsible for 2'-O-methylation of tRNAs and rRNAs. The archaeal box C/D small RNP complex requires a small RNA component (sRNA) possessing Watson-Crick complementarity to the target RNA along with three proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from S-adenosylmethionine to the target RNA is performed by fibrillarin, which by itself has no affinity for the sRNA-target duplex. Instead, it is targeted to the site of methylation through association with Nop5p, which in turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a bridge between the targeting and catalytic functions of the box C/D small RNP complex, we have employed alanine scanning to evaluate the interaction between the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA complex. From these data, we were able to construct an isolated RNA-binding domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography and binds to the L7Ae box C/D RNA complex with near wild type affinity. These data demonstrate that the Nop-RBD is an autonomously folding and functional module important for protein assembly in a number of complexes centered on the L7Ae-kinkturn RNP.
Highlights
Regions associated with function [8, 12, 13]
Box C/D ribonucleoprotein (RNP)3 complexes serve as RNA-guided site-specific 2Ј-O-methyltransferases in both archaea and eukaryotes [15, 16] where they are referred to as small RNP complexes and small nucleolar RNPs, respectively
Target RNA pairs with the small RNA component (sRNA) guide sequence and is methylated at the 2Ј-hydroxyl group of the nucleotide five bases upstream of either the D or DЈ box motif of the sRNA (Fig. 1, star) [17, 18]
Summary
Site-directed Mutagenesis, Expression, and Purification—Pyrococcus horikoshii aFib (GI 14590005) and L7Ae (GI 74407908) were cloned, expressed, and purified essentially as previously described [22]. Electrophoretic Mobility Shift Assays—Assays to measure the apparent equilibrium dissociation constant (KD) of mNop5p alanine mutants and the Nop-RBD for the L7Ae box C/D RNA complex were performed with 12% acrylamide gels (acrylamide:bis-acrylamide, 29:1) as described previously [22]. Binding reactions for both mNop5p and Nop-RBD were conducted at 25 °C in a buffer containing 16 mM Kϩ-HEPES, pH 7.5, 80 mM KCl, 0.01% Nonidet P-40, 25 g/ml tRNA, and 2 nM labeled RNA. In calculating the KD for each mutant, we assumed that each protein was 100% active
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