Four small RNA self‐cleaving domains, the hammerhead, hairpin, hepatitis delta virus and Neurospora VS ribozymes, have been identified previously in naturally occurring RNAs. The secondary structures of these ribozymes are reasonably well understood, but little is known about long‐range interactions that form the catalytically active tertiary conformations. Our previous work, which identified several secondary structure elements of the VS ribozyme, also showed that many additional bases were protected by magnesium‐dependent interactions, implying that several tertiary contacts remained to be identified. Here we have used site‐directed mutagenesis and chemical modification to characterize the first long‐range interaction identified in VS RNA. This interaction contains a 3 bp pseudoknot helix that is required for tertiary folding and self‐cleavage activity of the VS ribozyme.

Neurospora VS RNA performs an RNAmediated self-cleavage reaction whose products contain 2',3'-cyclic phosphate and 5'-hydroxyl termini. This reaction is similar to those of hammerhead, hairpin, and hepatitis 8 virus ribozymes; however, VS RNA is not similar in sequence to these other self-cleaving motifs. Here we propose a model for the secondary structure of the self-cleaving region ofVS

We have investigated the self-cleavage reaction performed by a ribozyme that contains 164 nucleotides of Neurospora VS RNA. Self-cleavage requires a divalent cation, magnesium being more effective than manganese or calcium. Spermidine or monovalent cations stimulate the reaction but cannot replace magnesium. The temperature optimum is rather broad, around 40-50 degrees C. Unlike some other ribozymes, VS self-cleavage is inhibited by even low concentrations of urea or formamide. The rate of cleavage is the same from pH 5.5 to 8.9, suggesting either that hydroxide is not directly involved in the cleavage chemistry or that a step that precedes the actual cleavage event is rate-limiting.

Most of the small ribozymes, including those that have been investigated as potential therapeutic agents, appear to be rather poor catalysts. These RNAs use an internal phosphoester transfer mechanism to catalyze site-specific RNA cleavage with apparent cleavage rate constants typically <2 min ؊1 . We have identified variants of one of these, the Neurospora Varkud satellite ribozyme, that self-cleaves with experimentally measured apparent rate constants of up to 10 s ؊1 (600 min ؊1 ), Ϸ2 orders of magnitude faster than any previously characterized self-cleaving RNA. We describe structural features of the cleavage site loop and an adjacent helix that affect the apparent rate constants for cleavage and ligation and the equilibrium between them. These data show that the phosphoester transfer ribozymes can catalyze reactions with rate constants much larger than previously appreciated and in the range of those of protein enzymes that perform similar reactions. S equence-or structure-specific cleavage of RNA phosphodiester bonds by many protein enzymes is quite rapid: for example, ribonuclease III cleaves its target RNA structure with an apparent rate constant (k obs ) of 6.4 s Ϫ1, and RNaseA can cleave its preferred dinucleotide sequence even faster, from 15.2 to 675 s Ϫ1 , depending on the source of the enzyme (1, 2). Site-specific hydrolytic cleavage of RNA by the RNA subunit of Bacillus RNaseP or the Tetrahymena self-splicing group I intron has been observed (3) or calculated (4) to be fast, in the range of 6 s Ϫ1 . A rate constant of Ϸ10 s Ϫ1was measured for a ligase ribozyme obtained by in vitro selection to catalyze the attack of a 3Ј hydroxyl on a 5Ј triphosphate (5).In contrast, most ribozymes appear to be rather poor catalysts. The ''small ribozymes,'' comprising the naturally occurring hammerhead, hairpin, hepatitis delta virus, and Neurospora Varkud satellite (VS) ribozymes, catalyze a transesterification reaction, yielding cleavage products with 2Ј3Ј cyclic phosphate and 5Ј hydroxyl termini like those produced by many protein ribonucleases. The vast majority of ribozymes selected in vitro to cleave RNA phosphodiester bonds also use this same phosphoester transfer chemistry and, like their natural counterparts, have cleavage rate constants of ϽϷ2 min Ϫ1 (0.033 s Ϫ1 ) (6, 7). A variety of enzymological considerations that affect ribozyme reaction rates have been discussed (8), and it has been recently proposed that chemical principles may limit the rates of certain small ribozymes (9, 10).The VS ribozyme is found in RNA transcripts of a plasmid in the mitochondria of certain natural isolates of the fungus Neurospora (11). It catalyzes site-specific cleavage and ligation reactions, similar to those performed by hammerhead, hairpin, and hepatitis delta virus ribozymes that are involved in the replication of the RNAs that contain the ribozyme (reviewed in refs. 12 and 13). Cleavage in VS RNA occurs after nucleotide G620 in an internal loop between helices Ia and Ib (Fig. 1B) (14). Biophysical, crosslinking, mu...