Loop dependence of the stability and dynamics of nucleic acid hairpins

Hairpin loops are critical to the formation of nucleic acid secondary structure, and to their function. Previous studies revealed a steep dependence of single-stranded DNA (ssDNA) hairpin stability with length of the loop (L) as ∼L8.5 ± 0.5, in 100 mM NaCl, which was attributed to intraloop stacking interactions. In this article, the loop-size dependence of RNA hairpin stabilities and their folding/unfolding kinetics were monitored with laser temperature-jump spectroscopy. Our results suggest that similar mechanisms stabilize small ssDNA and RNA loops, and show that salt contributes significantly to the dependence of hairpin stability on loop size. In 2.5 mM MgCl2, the stabilities of both ssDNA and RNA hairpins scale as ∼L4 ± 0.5, indicating that the intraloop interactions are weaker in the presence of Mg2+. Interestingly, the folding times for ssDNA hairpins (in 100 mM NaCl) and RNA hairpins (in 2.5 mM MgCl2) are similar despite differences in the salt conditions and the stem sequence, and increase similarly with loop size, ∼L2.2 ± 0.5 and ∼L2.6 ± 0.5, respectively. These results suggest that hairpins with small loops may be specifically stabilized by interactions of the Na+ ions with the loops. The results also reinforce the idea that folding times are dominated by an entropic search for the correct nucleating conformation

Rapid binding and release of Hfq from ternary complexes during RNA annealing

The Sm protein Hfq binds small non-coding RNA (sRNAs) in bacteria and facilitates their base pairing with mRNA targets. Molecular beacons and a 16 nt RNA derived from the Hfq binding site in DsrA sRNA were used to investigate how Hfq accelerates base pairing between complementary strands of RNA. Stopped-flow fluorescence experiments showed that annealing became faster with Hfq concentration but was impaired by mutations in RNA binding sites on either face of the Hfq ring or by competition with excess RNA substrate. A fast bimolecular Hfq binding step (∼108 M−1s−1) observed with Cy3-Hfq was followed by a slow transition (0.5 s−1) to a stable Hfq–RNA complex that exchanges RNA ligands more slowly. Release of Hfq upon addition of complementary RNA was faster than duplex formation, suggesting that the nucleic acid strands dissociate from Hfq before base pairing is complete. A working model is presented in which rapid co-binding and release of two RNA strands from the Hfq ternary complex accelerates helix initiation 10 000 times above the Hfq-independent rate. Thus, Hfq acts to overcome barriers to helix initiation, but the net reaction flux depends on how tightly Hfq binds the reactants and products and the potential for unproductive binding interactions

A minimized rRNA-binding site for ribosomal protein S4 and its implications for 30S assembly

Primary ribosomal protein S4 is essential for 30S ribosome biogenesis in eubacteria, because it nucleates subunit assembly and helps coordinate assembly with the synthesis of its rRNA and protein components. S4 binds a five-helix junction (5WJ) that bridges the 5′ and 3′ ends of the 16S 5′ domain. To delineate which nucleotides contribute to S4 recognition, sequential deletions of the 16S 5′ domain were tested in competitive S4-binding assays based on electrophoretic mobility shifts. S4 binds the minimal 5WJ RNA containing just the five-helix junction as well or better than with affinity comparable to or better than the 5′ domain or native 16S rRNA. Internal deletions and point mutations demonstrated that helices 3, 4, 16 and residues at the helix junctions are necessary for S4 binding, while the conserved helix 18 pseudoknot is dispensable. Hydroxyl radical footprinting and chemical base modification showed that S4 makes the same interactions with minimal rRNA substrates as with the native 16S rRNA, but the minimal substrates are more pre-organized for binding S4. Together, these results suggest that favorable interactions with S4 offset the energetic penalty for folding the 16S rRNA

Global Stabilization of rRNA Structure by Ribosomal Proteins S4, S17, and S20

U radu se analiziraju rezultati longitudinalnih istraživanja o potrošnji kućanstva za Republiku Hrvatsku na temelju anketa dostupnih na stranicama Državnog zavoda za statistiku. Rezultati za Republiku Hrvatsku uspoređuju se s rezultatima odabranih zemalja koje su provodile sličnu anketu i čiji rezultati se mogu pronaći na stranicama Eurostat-a

RNA compaction and iterative scanning for small RNA targets by the Hfq chaperone

Abstract RNA-guided enzymes must quickly search a vast sequence space for their targets. This search is aided by chaperones such as Hfq, a protein that mediates regulation by bacterial small RNAs (sRNAs). How RNA binding proteins enhance this search is little known. Using single-molecule Förster resonance energy transfer, we show that E. coli Hfq performs a one-dimensional scan in which compaction of the target RNA delivers sRNAs to sites distant from the location of Hfq recruitment. We also show that Hfq can transfer an sRNA between different target sites in a single mRNA, favoring the most stable duplex. We propose that compaction and segmental transfer, combined with repeated cycles of base pairing, enable the kinetic selection of optimal sRNA targets. Finally, we show that RNA compaction and sRNA transfer require conserved arginine patches. We suggest that arginine patches are a widespread strategy for enabling the movement of RNA across protein surfaces