No abstract

Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two threeway junctions. Second, catalytic activity of the ribozyme is essential for replication of VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that the VS ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS ribozyme favor cleavage of the substrate downstream of the ribozyme core instead. This preference is expected to facilitate processing of the multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.

Covalent dimers of alamethicin form conducting structures with gating properties that permit measurement of current-voltage (I-V) relationships during the lifetime of a single channel. These I-V curves demonstrate that the alamethicin channel is a rectifier that passes current preferentially, with voltages of the same sign as that of the voltage that induced opening of the channel. The degree of rectification depends on the salt concentration; single-channel I-V relationships become almost linear in 3 M potassium chloride. These properties may be qualitatively understood by using Poisson-Nernst-Planck theory and a modeled structure of the alamethicin pore.

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...

RNA editing by members of the ADAR (adenosine deaminase that acts on RNA) enzyme family involves hydrolytic deamination of adenosine to inosine within the context of a double-stranded pre-mRNA substrate. Editing of the human GluR-B transcript is catalyzed by the enzyme ADAR2 at the Q/R and R/G sites. We have established a minimal RNA substrate for editing based on the R/G site and have characterized the interaction of ADAR2 with this RNA by gel shift, kinetic, and crosslinking analyses. Gel shift analysis revealed that two complexes are formed on the RNA as protein concentration is increased; the ADAR monomers can be crosslinked to one another in an RNA-dependent fashion. We performed a detailed kinetic study of the editing reaction; the data from this study are consistent with a reaction scheme in which formation of an ADAR2⅐RNA ternary complex is required for efficient RNA editing and in which formation of this complex is rate determining. These observations suggest that RNA adenosine deaminases function as homodimers on their RNA substrates and may partially explain regulation of RNA editing in these systems.Eukaryotic precursor-mRNAs (pre-mRNAs) are subject to a variety of post-transcriptional modifications that regulate gene expression. These modifications, collectively referred to as RNA processing reactions, include 5Ј-and 3Ј-end formation, RNA splicing, polyadenylation, and the recently characterized phenomenon of RNA editing (reviewed in Refs.

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.

Single-channel measurements were used to study the cis-trans isomerization of a carbamate bond in gramicidin-ethylenediamine, a derivative of the channel-forming peptide gramicidin. From the temperature dependence of transition rates, values of 64.9 f 2.5 and 58.2 f 3.0 kJ mol-' were calculated for isomerization from the trans and cis states, respectively. The entropies of activation were calculated to be

Alamethicin channels are prototypical helix bundles that may serve as tractable models for more complex protein ion channels. Solid-phase peptide synthesis of alamethicin analogues using FMOC-amino acid fluorides followed by chemical dimerization of these peptides facilitates structure-function studies of particular channel states in bilayer membranes. State 3 in particular, tentatively assigned to a hexameric helix bundle, is sufficiently long-lived that current-voltage measurements can be made during the lifetime of an individual channel opening. Molecular models of hexameric helix bundles, generated using restrained molecular dynamics with simulated annealing, indicate that a Gln7-->Asn7 (Q7-->N7) mutation will increase channel diameter locally. Experimentally, the conductance of state 3 of the N7-alm channel is found to be larger than that of the Q7-alm channel when ion flow is in the usual direction (cations entering the C-terminal end of the channel). When ion flow is in the opposite direction, no difference in the conductances of state 3 of Q7 and state 3 of N7 channels is observed. These results indicate that the effect of a change in pore diameter at position 7 is dependent on the magnitude of other barriers to permeation and that these barriers are voltage-dependent.