A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

Abstract

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the bindingcompetent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5 ′ -splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5 ′ -exon intermediate in self splicing to remain bound subsequent to 5 ′ -exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs.

Slow molecular recognition by RNA

“…When else might RNA prove advantageous? Folding studies have demonstrated that RNA readily adopts stable alternative structures-and we and others have argued and shown that these alternative structures can be inactive and deleterious (Gartland and Sueoka 1966;Lindahl et al 1966;Herschlag 1995;Uhlenbeck 1995;Russell 2008); however, riboswitches are biological RNAs that use such metasta- Finally, the slow observed ligand recognition by RNA provides an opportunity for kinetic control of specificity when binding is rate limiting (Karbstein and Herschlag 2003;Gleitsman and Herschlag 2014). In this mechanism, referred to as "k on specificity," added neighboring interactions can increase the lifetime of an initial encounter complex and thereby provide an attached ligand multiple chances at productive binding prior to diffusing away and thus a higher k on .…”

Nucleobases Undergo Dynamic Rearrangements during RNA Tertiary Folding

“…RNA folding can be initiated by introduction of specific ions (typically Mg 2+ ), then the temporal progress of chemical probing of the phosphodiester backbone is monitored in quenched-flow experiments. Timescales and intermediates during tertiary structure formation have been identified for the 414 nucleotide Tetrahymena Group I intron[14–19],[20], and the 215 nucleotide Azoarcus Group I intron[21–24]. Folding of the Tetrahymena Group I intron has been mapped by time-resolved hydroxyl radical footprinting, revealing a hierarchical folding pathway[25,26].…”

Interactions between the Azoarcus Ribozyme and Manganese (II) Divalent Cations

“…Previous attempts to perform catalytic and self assembly reactions with Azoarcus in the absence of Mg 2+ have failed or shown negligent yields [18]. Here we show that Mn 2+ can enhance catalytic capabilities of the Azoarcus ribozyme in a trans configuration, PUTT reaction, and we speculate on how the tertiary structure of Azoarcus affects cofactor compatibility according to previous research [19]. With evidence that manganese (II) ions were able to support the catalytic activity of the Azoarcus ribozyme , efforts were taken to understand the operational boundaries of manganese based PUTT reactions.…”

“…M1-M2, M5, and M12 were identified as the core metals, and make contact directly with the scaffold, while M8-M11 and M14-M16 also bind to P4. Other metal groups in the surrounding regions were found to be essential for structural stability [18,19]. Previous metal rescue experiments with Azoarcus have been attempted in the past using Ca 2+…”