RNA and the Goldilocks Zone: Where Mg2+ concentration is just right

Mg2+, the most abundant divalent cation in cells, catalyzes RNA cleavage but can also promote RNA folding. Because folding can protect RNA from cleavage, we predicted a "Goldilocks zone", which is a local maximum in RNA lifetime at the minimum Mg2+ concentration required for folding. By simulation and experiment, we characterized the RNA Goldilocks zone and its dependence on cleavage parameters and extent of folding. We show experimentally that yeast tRNAPhe can inhabit a Goldilocks zone. The Goldilocks phenomena appears to be robust and is tunable by changes in magnesium affinity, and a variety of other factors. Goldilocks behavior can be more pronounced for RNAs with intermediate folding states. Goldilocks behavior allows ultrafine control of RNA chemical lifetime. A subset of RNAs in vivo are expected to occupy the Goldilocks zone. In evolutionary context, Goldilocks behavior may have shaped RNA in an early Earth environment containing Mg2+ and other metals. RNAs that do not fold cannot access a Goldilocks zone.

Time-resolved NMR monitoring of tRNA maturation

ABSTRACTAlthough the biological importance of post-transcriptional RNA modifications in gene expression is widely appreciated, methods to directly detect the introduction of these modifications during RNA biosynthesis are rare and do not easily provide information on the temporal nature of events. Here we introduce the application of NMR spectroscopy to observe the maturation of tRNAs in cell extracts. By following the maturation of yeast tRNAPhewith time-resolved NMR measurements, we found that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. In particular, we uncovered a strong hierarchy in the introduction of the T54, Ψ55 and m1A58 modifications in the T-arm, and demonstrate that the modification circuits identified in yeast extract with NMR also impact the tRNA modification process in living cells. The NMR-based methodology presented here could be adapted to investigate different aspects of tRNA maturation and RNA modifications in general.

Sodium chloride-induced conformational change in tRNA as measured by circular dichroism

The effect of 0.01-1 M sodium ions on the conformation of the folded brewer’s yeast tRNAPhe was examined by circular dichroism method in the region 200-350 nm. The minimum peak at about 210 nm for tRNA solution with 50 mM sodium chloride showed a decrease in magnitude by 26-30% in comparison to that recorded for the solution of higher NaCl content. The depths of the peaks at 225 nm and 233 nm for two solutions with the lowest sodium chloride concentrations (cNaCl = 10mM, cNaCl = 50mM) were changed by 3-10% relative to the those in the spectra of other samples, for the 260 nm maximum peak a decrease in height was 21-25%. In the region 300-350 nm no significant difference was observed. The results point to a strong relationship between concentration of sodium ions and stabilization process of secondary and tertiary tRNA structure, which indicates the influence of sodium ions on stacking and base-pairing interactions.