Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution.

The interstitial liquid phase within frozen aqueous solutions is an environment that minimizes RNA degradation and facilitates reactions that may have relevance to the RNA World hypothesis. Previous work has shown that frozen solutions support condensation of activated nucleotides into RNA oligomers, RNA ligation by the hairpin ribozyme, and RNA synthesis by a RNA polymerase ribozyme. In the current study, we examined the activity of a hammerhead ribozyme (HHR) in frozen solution. The Schistosoma mansoni hammerhead ribozyme, which predominantly cleaves RNA, can ligate its cleaved products (P1 and P2) with yields up to ~23 % in single turnover experiments at 25 °C in the presence of Mg(2+). Our studies show that this HHR ligates RNA oligomers in frozen solution in the absence of divalent cations. Citrate and other anions that exhibit strong ion-water affinity enhanced ligation. Yields up to 43 % were observed in one freeze-thaw cycle and a maximum of 60 % was obtained after several freeze-thaw cycles using wild-type P1 and P2. Truncated and mutated P1 substrates were ligated to P2 with yields of 14-24 % in one freeze-thaw cycle. A pool of P2 substrates with mixtures of all four bases at five positions were ligated with P1 in frozen solution. High-throughput sequencing indicated that 70 of the 1024 possible P2 sequences were represented in ligated products at 1000 or more read counts per million reads. The results indicate that the HHR can ligate a range of short RNA oligomers into an ensemble of diverse sequences in ice.

Molecular paleontology: a biochemical model of the ancestral ribosome.

Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.

RNA folding and catalysis mediated by iron (II).

Mg²âº shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe²âº in the absence of free oxygen as a replacement for Mg²âº in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg²âº in RNA folding and function can indeed be served by Fe²âº. The results of quantum mechanical calculations show that the geometry of coordination of Fe²âº by RNA phosphates is similar to that of Mg²âº. Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4-P6 domain RNA is conserved between complexes with Fe²âº or Mg²âº. The catalytic activities of both the L1 ribozyme ligase, obtained previously by in vitro selection in the presence of Mg²âº, and the hammerhead ribozyme are enhanced in the presence of Fe²âº compared to Mg²âº. All chemical footprinting and ribozyme assays in the presence of Fe²âº were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe²âº. The combined biochemical and paleogeological data are consistent with a role for Fe²âº in an RNA World. RNA and Fe²âº could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg²âº alone.