Mg2+-Induced Compaction of Single RNA Molecules Monitored by Tethered Particle Microscopy

Abstract

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 nonspecific tethering, we have tracked the nanometer-scale Brownian motion of RNA-tethered fluorescent microspheres in all three dimensions at pH 7.5, 22 degrees C, in 10 mM or 100 mM NaCl in the absence or presence of 10 mM MgCl(2). The addition of Mg(2+) 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 Mg(2+) by a statistical Gaussian polymer model. We attribute these differences to the Mg(2+)-induced compaction of the relatively weakly structured and structurally disperse poly(U) mRNA, in contrast to Mg(2+)-induced reinforcement of existing secondary and tertiary structure contacts in the highly structured 16S rRNA. Both effects are nonspecific, however, as they are dampened in the presence of higher concentrations of monovalent cations.

Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

“…On this normalized scale, 1M7 reacted overall slightly more strongly than Tb 3+ , giving an average reactivity along the mRNA of ∼0.43 ± 0.12 compared with ∼0.35 ± 0.06 for Tb 3+ . Although addition of 1 mM Tb 3+ to probing reactions altered the ionic strength by <3%, ions of greater charge density, such as Tb 3+ , can preferentially stabilize structural elements (including tertiary structures) in an RNA by shielding its negatively charged backbone (Walter et al 2000;Walter 2003, 2005;Jeong et al 2003;Harris et al 2004;Newby Lambert et al 2006;Sefcikova et al 2007b). This feature is a plausible cause for the on average lower overall reactivity of gene 60 mRNA to Tb 3+ ions than to the neutral SHAPE reagent.…”

Tethered particle motion mediated by scattering from gold nanoparticles and darkfield microscopy

“…They utilized ∅ = 40 nm gold particles as reporters and studied the transcription of RNA [13]. More publications followed in which different enzymes were studied by means of video-enhanced DIC microscopy, TIRF microscopy, fluorescence microscopy or conventional transmission microscopy [14][15][16][17][18][19][20][21]. In all of these studies rather large polystyrene/latex particles (with diameters between 200 and 1000 nm) were applied.…”

A new optical method for characterizing single molecule interactions based on dark field microscopy

“…Mono-and multivalent cations are known to compact DNA and RNA structure [20][21][22] . Various techniques have been used to study the effect of cations on the conformation of DNA and RNA.…”