RNA molecules are exquisitely sensitive to the properties of counterions. The folding equilibrium of the Tetrahymena ribozyme is measured by non-denaturing gel electrophoresis in the presence of divalent group IIA metal cations. The stability of the folded ribozyme increases with the charge density (ζ) of the cation. Similar scaling is found when the free energy of the RNA folded in small and large metal cations is measured by urea denaturation. Brownian dynamics simulations of a polyelectrolyte show that the experimental observations can be explained by non-specific ion-RNA interactions in the absence of site-specific metal chelation. The experimental and simulation results establish that RNA stability is largely determined by a combination of counterion charge and the packing efficiency of condensed cations that depends on the excluded volume of the cations.

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Folding mechanisms in which secondary structures are stabilized through the formation of tertiary interactions are well documented in protein folding but challenge the folding hierarchy normally assumed for RNA. However, it is increasingly clear that RNA could fold by a similar mechanism. P5abc, a small independently folding tertiary domain of the Tetrahymena thermophila group I ribozyme, is known to fold by a secondary structure rearrangement involving helix P5c. However, the extent of this rearrangement and the precise stage of folding that triggers it are unknown. We use experiments and simulations to show that the P5c helix switches to the native secondary structure late in the folding pathway and is directly coupled to the formation of tertiary interactions in the A-rich bulge. P5c mutations show that the switch in P5c is not rate-determining and suggest that non-native interactions in P5c aid folding rather than impede it. Our study illustrates that despite significant differences in the building blocks of proteins and RNA, there may be common ways in which they self-assemble.

DbpA is a DEAD-box RNA helicase implicated in Escherichia coli large ribosomal subunit assembly. Previous studies have shown that when the ATPase and helicase inactive DbpA construct, R331A, is expressed in E. coli cells, a large ribosomal subunit intermediate accumulates. The large subunit intermediate migrates as a 45S particle in a sucrose gradient. Here, using a number of structural and fluorescent assays, we investigate the ribosome profiles of cells lacking wild-type DbpA and overexpressing the R331A DbpA construct. Our data show that in addition to the 45S particle previously described, 27S and 35S particles are also present in the ribosome profiles of cells overexpressing R331A DbpA. The 27S, 35S, and 45S independently convert to the 50S subunit, suggesting that ribosome assembly in the presence of R331A and the absence of wild-type DbpA occurs via multiple pathways.

23S ribosomal RNA (rRNA) of Escherichia coli 50S large ribosome subunit contains 26 post-transcriptionally modified nucleosides. Here, we determine the extent of modifications in the 35S and 45S large subunit intermediates, accumulating in cells expressing the helicase inactive DbpA protein, R331A, and the native 50S large subunit. The modifications we characterized are 3-methylpseudouridine, 2-methyladenine, 5-hydroxycytidine, and nine pseudouridines. These modifications were detected using 1-cyclohexyl-3-(2-morpholinoethyl)­carbodiimide metho-p-toluenesulfonate (CMCT) treatment followed by alkaline treatment. In addition, KMnO4 treatment of 23S rRNA was employed to detect 5-hydroxycytidine modification. CMCT and KMnO4 treatments produce chemical changes in modified nucleotides that cause reverse transcriptase misincorporations and deletions, which were detected employing next-generation sequencing. Our results show that the 2-methyladenine modification and seven uridines to pseudouridine isomerizations are present in both the 35S and 45S to similar extents as in the 50S. Hence, the enzymes that perform these modifications, namely, RluA, RluB, RluC, RluE, RluF, and RlmN, have already acted in the intermediates. Two uridines to pseudouridine isomerizations, the 3-methylpseudouridine and 5-hydroxycytidine modifications, are significantly less present in the 35S and 45S, as compared to the 50S. Therefore, the enzymes that incorporate these modifications, RluD, RlmH, and RlhA, are in the process of modifying the 35S and 45S or will incorporate these modifications during the later stages of ribosome assembly. Our study employs a novel high throughput and single nucleotide resolution technique for the detection of 2-methyladenine and two novel high throughput and single nucleotide resolution techniques for the detection of 5-hydroxycytidine.

Polyacrylamide gel electrophoresis under native conditions (native PAGE) is a well-established and versatile method for probing nucleic acid conformation and nucleic acid-protein interactions. Native PAGE has been used to measure RNA folding equilibria and kinetics under a wide variety of conditions. Advantages of this method are its adaptability, absolute determination of reaction endpoints, and direct analysis of conformational hetereogeneity within a sample. Native PAGE is also useful for resolving ligand-induced structural changes.

DbpA is a DEAD-box RNA helicase implicated in the assembly of the large ribosomal subunit. Similar to all the members of the DEAD-box family, the DbpA protein has two N-terminal RecA-like domains, which perform the RNA unwinding. However, unlike other members of this family, the DbpA protein also possesses a structured C-terminal RNA-binding domain that mediates specific tethering of DbpA to hairpin 92 of the Escherichia coli 23S ribosomal RNA. Previous studies using model RNA molecules containing hairpin 92 show that the RNA molecules support the DbpA protein's double-helix unwinding activity, provided that the double helix has a 3 ′ single-stranded region. The 3 ′ single-stranded region was suggested to be the start site of the DbpA protein's catalytic unwinding activity. The data presented here demonstrate that the single-stranded region 3′ of the doublehelix substrate is not required for the DbpA protein's unwinding activity and the DbpA protein unwinds the double-helix substrates by directly loading on them.