RNA folding in crowded solutions
Duncan Kilburn (Johns Hopkins University)
RNA molecules must fold to form their biologically active native state in the crowded environment of real cells. Most folding experiments, however, are carried out in dilute solution, and so require corrections to their outcomes to be applicable in vivo. We are studying RNA structure and folding in crowded solutions to empirically determine principles for such corrections, and therefore determine the true stability of folded RNA molecules in the cellular milieu.
We used small angle x-ray scattering experiments to measure scattering functions of a 64 kDa bacterial group I ribozyme in the presence of stabilizing ions (MgCl2 and NaCl) and polyethylene-glycol with different molecular weights as crowders.
We find that crowder molecules favor more compact states of the unfolded RNA, and also stabilize the folded state with respect to the unfolded state by up to 5 kT, as measured via the lowering of the folding midpoint on both MgCl2 and NaCl titrations. Crowders are also observed to compress the native state of this ribozyme and we have measured a concurrent increase in ribozyme activity. These effects cannot be explained by changes in Mg2+ or water activity.
In addition, we compare the scattering functions of RNAs stabilized to the same Rg in solutions with different crowders. We observe lower intensity scattering at approx. Q > 0.08 Å -1 for RNA in higher ionic strength solutions, indicating lower electron density correlations within those RNA particles on length scales below 80 Å. From the scattering functions we calculate the correlation length per volume for the particles and show that crowders restrict the number of conformations accessible to the RNA molecule compared with RNA stabilized exclusively by ions.
Back to Seminar Home Page
Last modified 18-January-2012 by website owner: NCNR (attn: Yun Liu)