Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme

Abstract

Kissing interactions in RNA are formed when bases between two hairpin loops pair. Intra-and intermolecular kissing interactions are important in forming the tertiary or quaternary structure of many RNAs. Self-cleavage of the wild-type Varkud satellite (VS) ribozyme requires a kissing interaction between the hairpin loops of stemloops I and V. In addition, self-cleavage requires a rearrangement of several base pairs at the base of stem I. We show that the kissing interaction is necessary for the secondary structure rearrangement of wild-type stem-loop I. Surprisingly, isolated stem-loop V in the absence of the rest of the ribozyme is sufficient to rearrange the secondary structure of isolated stem-loop I. In contrast to kissing interactions in other RNAs that are either confined to the loops or culminate in an extended intermolecular duplex, the VS kissing interaction causes changes in intramolecular base pairs within the target stem-loop.R NA kissing interactions, also called loop-loop pseudoknots, occur when the unpaired nucleotides in one hairpin loop base pair with the unpaired nucleotides in another hairpin loop (1). When the hairpin loops are located on separate RNA molecules, their intermolecular interaction is called a kissing complex. These interactions generally form between stem-loops containing extensive complementarity; however, stable complexes have been observed containing only two intermolecular Watson-Crick base pairs (2).Intramolecular kissing interactions are observed in the native structures of a variety of RNAs including Varkud satellite (VS) RNA, tRNA, and group I introns (3-6). These kissing interactions contribute to the assembly and stabilization of their respective RNA structures by joining and orienting helices. Kissing interactions may stabilize both native and nonnative interactions during tertiary folding, which can affect the rate at which the native structure is formed (7). Kissing interactions often form distorted structures that can serve as recognition sites for proteins, RNAs, metal ions, or other ligands (8-10). As a result, kissing interactions contribute to the stability of an RNA structure by affecting both global and local RNA interactions.The transient formation of an intermolecular kissing complex is required for RNA dimerization during the life cycle of retroviruses (11-15), and for the formation of some antisensetarget complexes (16,17). Kissing complexes in which the loop nucleotides are complementary can form stable dimers that contain intermolecular base pairs between the loop nucleotides only. Other stem-loops with more extensive complementarity sometimes form unstable kissing complexes, which are quickly remodeled into stable duplex or cruciform isoforms (18)(19)(20). Secondary structure remodeling involves breaking intramolecular base pairs in each hairpin and using the bases to form intermolecular helices.The VS ribozyme contains a kissing interaction that is required for self-cleavage of the wild-type RNA in vitro (6). This interaction involves Watson-Crick bas...

The Varkud satellite ribozyme

“…Both studies found that the internal loop comprised two sheared G·A base pairs reminiscent of domain 2 of the hammerhead ribozyme (Pley et al 1994;Scott et al 1995) plus a protonated A + ·C base pair. It has been proposed, on the basis of covariation analysis, that the loop-loop interaction between the substrate and stem V results in a rearrangement of the base pairing in stem Ib of the substrate (Andersen and Collins 2000). This involves the opening of the terminal base pair, the flipping out of C634, and rearrangement of base pairing in stem Ib, such that CCC (635-637) pairs with GGG (623-625).…”

“…On interacting with stem-loop V, C634 exhibits enhanced reactivity to dimethyl sulphate (Andersen and Collins 2001), but this might result from less than total extrusion of the base from the helix. A number of sequence changes that would be expected to stabilize the altered pattern of base pairing in helix Ib lead to a substantial reduction in cleavage rate (D. Lafontaine, A. McLeod, and D.M.J.…”

“…Part of this interaction is the formation of loop-loop pairing between the GAC triplet in the terminal loop of stem V, and a GUC triplet in the substrate (Rastogi et al 1996). The latter is largely contained within the terminal loop, but exposure of the 3Ј cytosine would require the opening of the terminal base pair, and it is proposed that this results in a change in the conformation of the substrate that is important for the cleavage reaction (Andersen and Collins 2000). In its trans-acting form, the ribozyme is very well behaved, especially if base pairing in helix Ia is absent (Fig.…”

Taming free energy landscapes with RNA chaperones

“…25 Examples of more dramatic secondary structure rearrangements that are coupled to tertiary folding transitions are found in the P5abc domain of the Tetrahymena group I ribozyme, 26 the Varkud satellite (VS) ribozyme, 27 and a pseudoknot in the E. coli α operon mRNA. 28 From these examples, it is apparent that the hierarchical levels of RNA structure are intimately linked with each other during the folding process.…”

Structure Probing of U4 snRNA: Investigations into the Mechanism of U4/U6 di-snRNP Formation.

“…Kissing loop complexes have been observed in a variety of RNAs such as tRNAs (Ladner et al 1975), and ribozymes (Lehnert et al 1996, Rastogi et al 1996, Anderson and Collins 2001, and are essential in biological processes including dimerization of retroviral RNAs Tinoco 2000, Reviewed in Paillart et al 1996). A well-known kissing loop complex controls replication of the ColE 1 plasmid in Escherichia coli.…”