Joan E. Olive

A long-range pseudoknot is required for activity of the Neurospora VS ribozyme.

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.

The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pK a

Several small ribozymes employ general acid-base catalysis as a mechanism to enhance site-specific RNA cleavage, even though the functional groups on the ribonucleoside building blocks of RNA have pK (a) values far removed from physiological pH. The rate of the cleavage reaction is strongly affected by the identity of the metal cation present in the reaction solution; however, the mechanism(s) by which different cations contribute to rate enhancement has not been determined. Using the Neurospora VS ribozyme, we provide evidence that different cations confer particular shifts in the apparent pK (a) values of the catalytic nucleobases, which in turn determines the fraction of RNA in the protonation state competent for general acid-base catalysis at a given pH, which determines the observed rate of the cleavage reaction. Despite large differences in observed rates of cleavage in different cations, mathematical models of general acid-base catalysis indicate that k (1), the intrinsic rate of the bond-breaking step, is essentially constant irrespective of the identity of the cation(s) in the reaction solution. Thus, in contrast to models that invoke unique roles for metal ions in ribozyme chemical mechanisms, we find that most, and possibly all, of the ion-specific rate enhancement in the VS ribozyme can be explained solely by the effect of the ions on nucleobase pK (a). The inference that k (1) is essentially constant suggests a resolution of the problem of kinetic ambiguity in favor of a model in which the lower pK (a) is that of the general acid and the higher pK (a) is that of the general base.

Enhancement of Neurospora VS ribozyme cleavage by tuberactinomycin antibiotics.

Several examples of inhibition of the function of a ribozyme or RNA‐protein complex have shown that certain antibiotics can interact specifically with RNA. There are, however, few examples of antibiotics that have a positive, rather than a negative, effect on the function of an RNA. We have found that micromolar concentrations of viomycin, a basic, cyclic peptide antibiotic of the tuberactinomycin group, enhance the cleavage of a ribozyme derived from Neurospora VS RNA. Viomycin decreases by an order of magnitude the concentration of magnesium required for cleavage. It also stimulates an otherwise insignificant transcleavage reaction by enhancing interactions between RNA molecules. The ability of viomycin to enhance some RNA‐mediated reactions but inhibit others, including translation and Group I intron splicing, demonstrates the potential for natural selection by small molecules during evolution in the ‘RNA world’ and may have broader implications with respect to ribozyme expression and activity in contemporary cells.

An important role of G638 in the cis-cleavage reaction of the Neurospora VS ribozyme revealed by a novel nucleotide analog incorporation method

We describe a chemical coupling procedure that allows joining of two RNAs, one of which contains a site-specific base analog substitution, in the absence of divalent ions. This method allows incorporation of nucleotide analogs at specific positions even into large, cis-cleaving ribozymes. Using this method we have studied the effects of substitution of G638 in the cleavage site loop of the VS ribozyme with a variety of purine analogs having different functional groups and pK(a) values. Cleavage rate versus pH profiles combined with kinetic solvent isotope experiments indicate an important role for G638 in proton transfer during the rate-limiting step of the cis-cleavage reaction.

Identification of Separate Structural Features That Affect Rate and Cation Concentration Dependence of Self-Cleavage by the Neurospora VS Ribozyme†

The cleavage site of the Neurospora VS ribozyme is located in an internal loop in a hairpin called stem-loop I. Stem-loop I undergoes a cation-dependent structural change to adopt a conformation, termed shifted, that is required for activity. Using site-directed mutagenesis and kinetic analyses, we show here that the insertion of a single-stranded linker between stem-loop I and the rest of the ribozyme increases the observed self-cleavage rate constant by 2 orders of magnitude without affecting the Mg(2+) requirement of the reaction. A distinct set of mutations that favors the formation of the shifted conformation of stem-loop I decreases the Mg(2+) requirement by an order of magnitude with little or no effect on the observed cleavage rate under standard reaction conditions. Similar trends were seen in reactions that contained Li(+) instead of Mg(2+). Mutants with lower ionic requirements also exhibited increased thermostability, providing evidence that the shifted conformation of stem-loop I favors the formation of the active conformation of the RNA. In natural, multimeric VS RNA, where a given ribozyme core is flanked by one copy of stem-loop I immediately upstream and another copy 0.7 kb downstream, cleavage at the downstream site is strongly preferred, providing evidence that separation of stem-loop I from the ribozyme core reflects the naturally evolved organization of the RNA.

Additional roles of a peripheral loop-loop interaction in the Neurospora VS ribozyme.

Many RNAs contain tertiary interactions that contribute to folding the RNA into its functional 3D structure. In the VS ribozyme, a tertiary loop–loop kissing interaction involving stem–loops I and V is also required to rearrange the secondary structure of stem–loop I such that nucleotides at the base of stem I, which contains the cleavage–ligation site, can adopt the conformation required for activity. In the current work, we have used mutants that constitutively adopt the catalytically permissive conformation to search for additional roles of the kissing interaction in vitro. Using mutations that disrupt or restore the kissing interaction, we find that the kissing interaction contributes ∼1000-fold enhancement to the rates of cleavage and ligation. Large Mg2+-dependent effects on equilibrium were also observed: in the presence of the kissing interaction cleavage is favored >10-fold at micromolar concentrations of Mg2+; whereas ligation is favored >10-fold at millimolar concentrations of Mg2+. In the absence of the kissing interaction cleavage exceeds ligation at all concentrations of Mg2+. These data provide evidence that the kissing interaction strongly affects the observed cleavage and ligation rate constants and the cleavage–ligation equilibrium of the ribozyme.

Revision of the nucleotide sequence and RNA splicing pathway of the Neurospora mitochondrial gene encoding ATPase subunit 6

Previous sequence analysis of the Neurospora oli2 (ATP6) mitochondrial gene suggested that, in addition to a typical Group-I intron, it contained an unusual, mostly-palindromic, 93-nucleotide intron. We report here revisions of the nucleotide sequence and analysis of the size and sequence of reverse-transcriptase PCR products that show: (1) the Group-I intron splice sites are located as predicted by previous DNA sequence analysis; (2) the putative 93-nt intron is not excised from the mature mRNA, and most of this sequence is actually in the 5′ untranslated region. We conclude that the Neurospora ATP6 gene contains only one intro. Analysis of the cDNA sequence also confirms the non-universal nature of the Neurospora mitochondrial genetic code: a TGA codon inferred from the DNA sequence is present as UGA in the mRNA. This provides direct evidence that this codon is not altered, for example by RNA editing, to conform to the universal code.