An Examination of Changes in the Florida Seabreeze Climatology

Florida summer precipitation patterns are in part governed by the dominance of different Seabreeze regimes. In this study, we are comparing 60 years of data against itself to see if there has been a significant trend in the tendencies of these regimes. Our goal is to understand if there has been a change, what might be causing them, and if there are better ways we can define synoptic scale atmospheric conditions to better understand and predict precipitation patterns in the Southeast US

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

Stabilization of Hfq-mediated translational repression by the co-repressor Crc in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) govern translation of numerous transcripts during carbon catabolite repression. Here, Crc was shown to enhance Hfq-mediated translational repression of several mRNAs. We have developed a single-molecule fluorescence assay to quantitatively assess the cooperation of Hfq and Crc to form a repressive complex on a RNA, encompassing the translation initiation region and the proximal coding sequence of the P. aeruginosa amiE gene. The presence of Crc did not change the amiE RNA-Hfq interaction lifetimes, whereas it changed the equilibrium towards more stable repressive complexes. This observation is in accord with Cryo-EM analyses, which showed an increased compactness of the repressive Hfq/Crc/RNA assemblies. These biophysical studies revealed how Crc protein kinetically stabilizes Hfq/RNA complexes, and how the two proteins together fold a large segment of the mRNA into a more compact translationally repressive structure. In fact, the presence of Crc resulted in stronger translational repression in vitro and in a significantly reduced half-life of the target amiE mRNA in vivo. Although Hfq is well-known to act with small regulatory RNAs, this study shows how Hfq can collaborate with another protein to down-regulate translation of mRNAs that become targets for the degradative machinery

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