Joyce E. Heckman

Functional involvement of G8 in the hairpin ribozyme cleavage mechanism

The catalytic determinants for the cleavage and ligation reactions mediated by the hairpin ribozyme are integral to the polyribonucleotide chain. We describe experiments that place G8, a critical guanosine, at the active site, and point to an essential role in catalysis. Cross‐linking and modeling show that formation of a catalytic complex is accompanied by a conformational change in which N1 and O6 of G8 become closely apposed to the scissile phosphodiester. UV cross‐linking, hydroxyl‐radical footprinting and native gel electrophoresis indicate that G8 variants inhibit the reaction at a step following domain association, and that the tertiary structure of the inactive complex is not measurably altered. Rate–pH profiles and fluorescence spectroscopy show that protonation at the N1 position of G8 is required for catalysis, and that modification of O6 can inhibit the reaction. Kinetic solvent isotope analysis suggests that two protons are transferred during the rate‐limiting step, consistent with rate‐limiting cleavage chemistry involving concerted deprotonation of the attacking 2′‐OH and protonation of the 5′‐O leaving group. We propose mechanistic models that are consistent with these data, including some that invoke a novel keto–enol tautomerization.

Cleavage of Highly Structured Viral RNA Molecules by Combinatorial Libraries of Hairpin Ribozymes THE MOST EFFECTIVE RIBOZYMES ARE NOT PREDICTED BY SUBSTRATE SELECTION RULES

Combinatorial libraries of hairpin ribozymes representing all possible cleavage specificities (>105) were used to evaluate all ribozyme cleavage sites within a large (4.2-kilobase) and highly structured viral mRNA, the 26 S subgenomic RNA of Sindbis virus. The combinatorial approach simultaneously accounts for target site structure and dynamics, together with ribozyme folding, and the sequences that result in a ribozyme-substrate complex with maximal activity. Primer extension was used to map and rank the relative activities of the ribozyme pool against individual sites and revealed two striking findings. First, only a small fraction of potential recognition sites are effectively cleaved (activity-selected sites). Second, nearly all of the most effectively cleaved sites deviated substantially from the established consensus selection rules for the hairpin ribozyme and were not predicted by examining the sequence, or through the use of computer-assisted predictions of RNA secondary structure. In vitro selection methods were used to isolate ribozymes with increased activity against substrates that deviate from the GUC consensus sequence. trans-Acting ribozymes targeting nine of the activity-selected sites were synthesized, together with ribozymes targeting four sites with a perfect match to the cleavage site consensus (sequence-selected sites). Activity-selected ribozymes have much higher cleavage activity against the long, structured RNA molecules than do sequence-selected ribozymes, although the latter are effective in cleaving oligoribonucleotides, as predicted. These results imply that, for Sindbis virus 26 S RNA, designing ribozymes based on matches to the consensus sequence may be an ineffective strategy.

Recent Developments in tRNA Sequencing Methods as Applied to Analyses of Mitochondrial tRNAs

With the extensive literature that has already accumulated on tRNAs, it may not be evident that this is a relatively new area of research. Discovered only about 22 years ago (Hoagland et al. 1957), following the prediction of the existence of molecules with similar properties by Crick, the first sequence of a tRNA and indeed of any nucleic acid was published only in 1965 (Holley et al. 1965). Since then the sequence of at least 120 different tRNAs from a variety of biological sources have been established (Sprinzl et al. 1978). The two factors that have contributed most to this rapid progress in tRNA sequencing have been the development (1) of column chromatographic and gel electrophoretic methods (Gillam et al. 1967; Cherayil and Bock 1965; Pearson et al. 1971; Ikemura and Dahlberg 1973) suitable for purification of tRNAs and (2) of rapid sequencing methods requiring only very small amounts of tRNAs. Thus, whereas the first sequence analysis of a tRNA, which utilized spectrophotometric procedures for identification of nucleotides (Holley 1968), required several hundred milligrams to a gram of purified tRNA and several years of effort, methods currently available enable one to sequence a tRNA with just a few micrograms (2–10 μ g) within a few weeks. The first few tRNAs to be sequenced were all from yeast (Holley et al. 1965; Zachau et al. 1966; Madison et al. 1966; RajBhandary et al. 1967b; Baev et al. 1967). To a large extent this was a reflection of yeast being a relatively...