Mg2+-Induced Compaction of Single RNA
Molecules
Monitored by Tethered Particle Microscopy
Meredith Newby Lambert 1, Eva Vocker 1, Stefanie Redemann 1, Seth Blumberg 1, Arivalagan Gajraj 1, Jens-Christian Meiners 1 and Nils G Walter 1*
1 University of Michigan
* To whom correspondence should be addressed. E-mail: nwalter{at}umich.edu.
Submitted on June 3, 2005
Revised on July 4, 2005
Accepted on 26 January 2006
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Abstract |
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We have applied tethered particle microscopy (TPM) as a single molecule analysis tool to studies of the conformational dynamics of poly-uridine(U) messenger (m)RNA and 16S ribosomal (r)RNA molecules. Using stroboscopic total internal reflection illumination and rigorous selection criteria to distinguish from non-specific tethering, we have tracked the nanometer-scale Brownian motion of RNA-tethered fluorescent microspheres in all three dimensions at pH 7.5, 22 oC, in 10 mM or 100 mM NaCl, in the absence or presence of 10 mM MgCl2. The addition of Mg2+ to low-ionic strength buffer results in significant compaction and stiffening of poly(U) mRNA, but not of 16S rRNA. Furthermore, the motion of poly(U)-tethered microspheres is more heterogeneous than that of 16S rRNA-tethered microspheres. Analysis of in-plane bead motion suggests that poly(U) RNA, but less so 16S rRNA, can be modeled both in the presence and absence of Mg2+ by a statistical Gaussian polymer model. We attribute these differences to the Mg2+-induced compaction of the relatively weakly structured and structurally disperse poly(U) mRNA, in contrast to Mg2+-induced reinforcement of existing secondary and tertiary structure contacts in the highly structured 16S rRNA. Both effects are non-specific, however, as they are dampened in the presence of higher concentrations of monovalent cations.
Key Words:
RNA folding, RNA structure, metal ion, ribosome, single-particle tracking, total internal reflection fluorescence microscopy
