College Park, Maryland      June 6 - 10 , 2004

WP8: Compaction of a Bacterial Group I Ribozyme by cations studied by SANS and SAXS

U.A. Perez-Salas (National Institute of Standards and Technology; University of California at Irvine), G.H. Caliskan (National Institute of Standards and Technology; Johns Hopkins University), P. Rangan (Johns Hopkins University; New York University), S Krueger (National Institute of Standards and Technology), R.M. Briber, D. Thirumalai (University of Maryland, College Park), S.A. Woodson (Johns Hopkins University)

Counterions are critical to the self-assembly of RNA tertiary structure because they neutralize the large electrostatic forces which oppose the folding process. Changes in the size and shape of the Azoarcus group I ribozyme as a function of Mg2+ and Na+ concentration were followed by small angle neutron scattering (SANS) experiments performed at the NIST Center for Neutron research. In low salt buffer, the RNA was expanded, with an average radius of gyration (Rg) of 53 1 . A highly cooperative transition to a compact form (Rg ) 31.5 0.5 was observed between 1.6 and 1.7 mM MgCl2. The collapse transition, which is unusually sharp in Mg2+, has the characteristics of a first-order phase transition. Partial digestion with ribonuclease T1 under identical conditions showed that this transition correlated with the assembly of double helices in the ribozyme core. Fivefold higher Mg2+ concentrations were required for self-splicing, indicating that compaction occurs before native tertiary interactions are fully stabilized. No further decrease in Rg was observed between 1.7 and 20 mM MgCl2, indicating that the intermediates have the same dimensions as the native ribozyme, within the uncertainty of the data (1 ). A more gradual transition to a final Rg of approximately 33.5 was observed between 0.45 and 2 M NaCl. This confirms the expectation that monovalent ions not only are less efficient in charge neutralization but also contract the RNA less efficiently than multivalent ions.

Complementary small angle x-ray scattering experiments were recently preformed at the Advanced Photon Source at Argonne National Laboratory on the Basic Energy Sciences Synchrotron Radiation Center (BESSRC) beamline and on the Biophysics Collaborative Access Team (Bio-CAT) . These preliminary experiments, which followed the size of the Azoarcus group I ribozyme as a function of the concentration of Na+, Mg2+, Ca2+ and Co3+ cations, show that the efficiency of charge neutralization increases with the increasing valence of the condensing cation. As observed in the SANS experiment, the collapse the ribozyme into a compact state does not coincide with self splicing activity yet the dimensions of the ribozyme in the compact state show no further decrease in Rg even after native conditions are reached. The compact intermediates in Na+, Ca2+ and Co3+, at their corresponding transition concentrations, show similar dimensions and only a couple of larger than the compact state in Mg2+. It is also observed that the transition to a collapsed state in Mg2+ and Ca2+ occurs at different cation concentrations, indicative of a cation size effect. In contrast with Na+, a ten-fold increase in Ca2+ beyond the collapse transition concentration shows the ribozyme compacting further to a dimension equivalent to the compact state in Mg2+. Although it is predicted that the Rg of the ribozyme is inversely proportional to the square of the cation valence, no further compactation of the ribozyme in Co3+ could be observed because it precipitated out of solution before this could occur.

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