NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site

Abstract

Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg 2؉ binding sites in the active internal loop. One of the Mg 2؉ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program MC-SEARCH, we identified the active internal loop fold in other RNA structures. In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme. RNA molecules play essential roles in many cellular processes. These include the enzymatic activity of ribozymes that are required for protein synthesis and certain RNA processing reactions (1). NMR and x-ray crystallographic studies have provided some insights into the relationship between RNA structure and catalysis; however, interpretation of structurefunction relationships, even in the well studied hammerhead ribozyme (2), continues to be challenging (1). It has also been difficult to make any generalizations about the role of RNA structure in catalysis, in part because of the small number of known ribozymes and the limited amount of structural information available. We are studying the Neurospora Varkud satellite (VS) ribozyme to provide information about the role of tertiary structure and conformational changes in RNA catalysis.The Neurospora VS ribozyme originates from an abundant RNA satellite of 881 nt found in the mitochondria of the Varkud-1c strain of Neurospora (3). Fragments of Ϸ120-180 nt derived from this natural RNA sequence undergo self-cleavage at a specific phosphodiester bond to produce 5Ј-OH and 2Ј,3Ј-cyclic phosphate termini (Fig. 1a) (3-5). Although these products are characteristic of other small ribozymes, the VS ribozyme possesses unique primary (4), secondary (6), and tertiary structures (7-9). The secondary structure of the self-cleaving VS ribozyme is characterized by six helical domains (Fig. 1a); stem-loop I forms the substrate domain and stem-loops II-VI comprise the catalytic domain (6). When these two domains are synthesized separately, the catalytic domain can perform the same cleavage reaction,...

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FR3D: finding local and composite recurrent structural motifs in RNA 3D structures

“…Fix a rotation matrix R and differentiate E(R, t) with respect to the components of t. Setting the derivatives equal to zero immediately leads to the unique solution (19) This is easier to understand if we express t * as t * = c̄ − R −1 b, where (20) are the weighted centers of mass of the two sets of vectors. Substituting this optimal value of t into the error E leaves…”

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