Exceptionally fast self-cleavage by a Neurospora Varkud satellite ribozyme

Abstract

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...

Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme

“…11. All reactions fit well to this analysis (R 2 Ն 0.99, and observed maximal extents of cleavage typically were 80-95%).…”

“…5) (11,39,41,45); the two mutational routes to shifting stem-loop I are thought to be functionally equivalent. Mutant RGkV disrupts the kissing interaction with three substitutions in loop V and was made in the context of the RG stem-loop I (11,39). Mutant A756C changes the putative active-site base A756 to C and is in the context of the CG stem-loop I (45).…”

“…42, are derivatives of RS19 (42) in which stem-loop I adopts the constitutively shifted conformation due to the presence of either a 634G substitution (mutant RG) or 624C ϩ 636G substitutions (mutant CG; Fig. 5) (11,39,41,45); the two mutational routes to shifting stem-loop I are thought to be functionally equivalent. Mutant RGkV disrupts the kissing interaction with three substitutions in loop V and was made in the context of the RG stem-loop I (11,39).…”

“…11, by using self-cleavage buffers (SCBs) at various pH levels in the presence of 200 mM MgCl 2 (chosen to be high enough that Mg 2ϩ binding would not limit the cleavage reaction). 1ϫ SCB contained 40 mM buffer (sodium acetate, pH 4.2-4.8; Mes, pH 4.6-5.8; sodium cacodylate, pH 6.0-6.8; sodium Hepes, pH 7.0-7.8; Tris⅐HCl, pH 8.0-8.8; and CAPSO, pH 9.0-9.6), 50 mM KCl, and 2 mM spermidine.…”

“…Recently, VS (11) and hammerhead (12, 13) ribozymes have been described that exhibit cleavage and/or ligation rate constants two to three orders of magnitude faster than observed with previous constructs, and in the range of those of protein enzymes that catalyze similar reactions. These RNAs appear to have overcome whatever slow step was limiting the observed rate of previous versions of the ribozyme and they may provide experimental systems to investigate faster steps in the kinetic pathway of site-specific phosphodiester bond cleavage.…”