Divalent ions tune the kinetics of a bacterial GTPase center rRNA folding transition from secondary to tertiary structure

Abstract

Folding of an RNA from secondary to tertiary structure often depends on divalent ions for efficient electrostatic charge screening (nonspecific association) or binding (specific association). To measure how different divalent cations modify folding kinetics of the 60 nucleotide E. coli rRNA GTPase center, we combined stopped-flow fluorescence in the presence of Mg 2+ , Ca 2+ , or Sr 2+ together with time-resolved small angle X-ray scattering (SAXS) in the presence of Mg 2+ to observe the folding process. Immediately upon addition of each divalent ion, the RNA undergoes a transition from an extended state with secondary structure to a more compact structure. Subsequently, specific divalent ions modulate populations of intermediates in conformational ensembles along the folding pathway with transition times longer than 10 msec. Rate constants for the five folding transitions act on timescales from submillisecond to tens of seconds. The sensitivity of RNA tertiary structure to divalent cation identity affects all but the fastest events in RNA folding, and allowed us to identify those states that prefer Mg 2+ . The GTPase center RNA appears to have optimized its folding trajectory to specifically utilize this most abundant intracellular divalent ion.

Ribosomal Protein L11 Selectively Stabilizes a Tertiary Structure of the GTPase Center rRNA Domain

“…In solution, thermodynamic studies of GAC folding showed that, in a background of KCl, divalent ions were necessary to stabilize the tertiary structure [17][18][19][20]. In our previous kinetic studies of Mg 2+ -dependent GAC tertiary folding, we found that the transition from secondary structure to tertiary structure has four intermediate states [21]. Our kinetic scheme for GAC folding includes two steps of divalent ion binding within the first 100 ms.…”

“…We needed to use several complementary methods to dissect the global and local responses of the GAC RNA to L11 CTD binding within our kinetic framework for GAC tertiary folding [21]. First, to determine the GAC tertiary fold in the absence of protein, we crystallized and determined the structure of the E. coli 58 nt domain.…”

“…To examine the global parameters of the RNA ensembles when bound by L11, we utilized small-angle X-ray scattering (SAXS). We previously found [21] that the scattering properties of the GAC RNA are sensitive to ion-induced folding: the GAC secondary structure has a calculated radius of gyration (Rg) of ~25 Å, while its folded tertiary structure has Rg = 21.3 ± 0.5 Å in the presence of Mg 2+ . Now we compare the dimensions of the GAC RNA when L11 CTD is bound, to measure what effect, if any, the protein has on the compaction of the RNA or the conformational ensemble.…”

Macromolecular crowding effects on transcription and translation are regulated by free magnesium ion

“…Mechanisms of Mg 2+ ‐regulated crowding effects on transcription and translation are still unclear. Recent studies on the effect of Mg 2+ on tRNA folding might provide some clues . Strulson et al.…”

Charge density of cation determines inner versus outer shell coordination to phosphate in RNA

“…As a consequence, ion-RNA interactions during the folding process has been the subject of extensive experimental and theoretical studies. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] From the theory of ion-induced shape changes in polyelectrolytes (PEs), it follows that divalent ions (such as Mg 2+ and Ca 2+ ) should be more efficient than monovalent ions in neutralizing the phosphate charges 30 in RNAs. 22,23 The physically appealing counterion condensation (CIC) theory could be used to estimate the fraction of condensed ions onto PEs with regular (rod-like or spherical) shape, and account for the phenomenon of entropically-driven counterion release in which monovalent ions are replaced by divalent ions.…”