48 research outputs found
A Guanosine-Centric Mechanism for RNA Chaperone Function
RNA chaperones are ubiquitous, heterogeneous proteins essential for RNA structural biogenesis and function. We investigated the mechanism of chaperone-mediated RNA folding by following the time-resolved dimerization of the packaging domain of a retroviral RNA at nucleotide resolution. In the absence of the nucleocapsid (NC) chaperone, dimerization proceeded via multiple, slow-folding intermediates. In the presence of NC, dimerization occurred rapidly via a single structural intermediate. The RNA binding domain of hnRNP A1 protein (UP1), a structurally unrelated chaperone, also accelerated dimerization. Both chaperones interacted primarily with guanosine residues. Replacing guanosine with more weakly pairing inosine yielded an RNA that folded rapidly without a facilitating chaperone. These results show RNA chaperones can simplify RNA folding landscapes by weakening intramolecular interactions involving guanosine and explain many RNA chaperone activities
Thermodynamic characterization of RNA triloops
ABSTRACT: Relatively few thermodynamic parameters are available for RNA triloops. Therefore, 24 stemloop sequences containing naturally occurring triloops were optically melted, and the thermodynamic parameters ΔH°, ΔS°, ΔG°3 7 , and T M for each stem-loop were determined. These new experimental values, on average, are 0.5 kcal/mol different from the values predicted for these triloops using the model proposed by Mathews et al. [Mathews, D. H., Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M., and Turner, D. H. (2004) Proc. Natl. Acad. Sci. U.S. A. 101, 7287-7292]. The data for the 24 triloops reported here were then combined with the data for five triloops that were published previously. A new model was derived to predict the free energy contribution of previously unmeasured triloops. The average absolute difference between the measured values and the values predicted using this proposed model is 0.3 kcal/mol. These new experimental data and updated predictive model allow for more accurate calculations of the free energy of RNA stemloops containing triloops and, furthermore, should allow for improved prediction of secondary structure from sequence. RNA stem-loops containing three nucleotides in the loop, triloops, are common secondary structure motifs found in naturally occurring RNA. For example, bacterial 16S rRNAs strongly favor tetraloops; however, the UUU triloop is the most common replacement (1). In the 16S-like rRNA variable regions, triloops account for 7% of the loops in bacteria and 16% of the loops in eukaryotes (2). Triloops are also found in large subunit rRNAs (3, 4), 5S rRNAs (5), signal recognition particles (6), RNase P RNAs (7), and group I introns (8, 9). More specifically, triloops are found in Brome mosaic virus (þ) strand RNA (10), human rhinovirus isotype 14 (11), iron responsive element RNA (12), and an RNA aptamer for bacteriophage MS2 coat protein (13), to name a few. Although relatively unstable due to the strain in the loop, triloops may be an important structural feature due to the accessibility of the loop nucleotides for recognition by proteins, other nucleic acids, or small molecules. It has been shown that triloops play a role in various biological processes, including virus replication The current model used by secondary structure prediction algorithms to predict the thermodynamic contribution of RNA triloops to stem-loop stability is sequence independent; all triloops contribute 5.4 kcal/mol to stem-loop stability, with the exception of 5 0 CCC3 0 which contributes 6.9 kcal/mol (21). In addition, there are two unstable triloop sequences (5 0 CAACG3 0 and 5 0 GUUAC3 0 ) for which this predictive model is not used; instead, the ΔG°3 7,loop values (6.8 and 6.9 kcal/mol, respectively) for these two triloops are provided in a lookup table (21). An interesting study by the Bevilacqua laboratory (19) used a combinatorial approach and temperature gradient gel electrophoresis to identify stable and unstable RNA triloops. It was discovered that sequence preferences for exceptionally stable triloops included a U-rich loop and C-G as the closing base pair. Although they used 10 mM NaCl during their melting experiments, they suggested that the rules for predicting triloop stability at 1 M NaCl should be modified; however, this has yet to be done. Here, we report the thermodynamic parameters for 24 previously unmeasured RNA triloops in 1 M NaCl and propose a new algorithm for predicting the contribution of triloops to stem-loop stability, which includes two bonuses for stabilizing sequence features. MATERIALS AND METHODS Compiling and Searching a Database for RNA Triloops. The initial aim of this project was to identify the most frequently occurring RNA triloops in nature and to thermodynamically characterize these hairpin triloop sequences. Therefore, a database of 1349 RNA secondary structures containing 123 small subunit rRNAs (22), 223 large subunit rRNAs (3, 4), 309 5S rRNAs (5), 484 tRNAs (23), 91 signal recognition particles (6), 16 RNase P RNAs (7), 100 group I introns (8, 9), and 3 group II introns (24) was compiled. This database was searched for triloops, and the number of occurrences for each type of triloop was tabulated. In this work, G-U pairs are considered to be canonical base pairs. Design of Sequences for Optical Melting Studies. Since most thermodynamic parameters for RNA secondary structure motifs are reported for RNA solutions containing 1 M NaCl, the melting buffer used in this work also contained 1 M NaCl. A major limitation of a thermodynamic analysis of RNA hairpins using this high salt concentration is the possible bimolecular
Energetic signatures of single base bulges: thermodynamic consequences and biological implications
DNA bulges are biologically consequential defects that can arise from template-primer misalignments during replication and pose challenges to the cellular DNA repair machinery. Calorimetric and spectroscopic characterizations of defect-containing duplexes reveal systematic patterns of sequence-context dependent bulge-induced destabilizations. These distinguishing energetic signatures are manifest in three coupled characteristics, namely: the magnitude of the bulge-induced duplex destabilization (ΔΔGBulge); the thermodynamic origins of ΔΔGBulge (i.e. enthalpic versus entropic); and, the cooperativity of the duplex melting transition (i.e. two-state versus non-two state). We find moderately destabilized duplexes undergo two-state dissociation and exhibit ΔΔGBulge values consistent with localized, nearest neighbor perturbations arising from unfavorable entropic contributions. Conversely, strongly destabilized duplexes melt in a non-two-state manner and exhibit ΔΔGBulge values consistent with perturbations exceeding nearest-neighbor expectations that are enthalpic in origin. Significantly, our data reveal an intriguing correlation in which the energetic impact of a single bulge base centered in one strand portends the impact of the corresponding complementary bulge base embedded in the opposite strand. We discuss potential correlations between these bulge-specific differential energetic profiles and their overall biological implications in terms of DNA recognition, repair and replication
New data on Caledonian, Alpine-style folding in the Holy Cross Mts., Poland
There has been a century-long debate on the nature of the major orogeny in the Holy Cross Mts. Some research workers consider that they were folded during the Variscan orogeny, and that Caledonian movements were responsible only for the formation of mesostructures. Others provide evidence for great folding movements and detachments, suggesting that strong Caledonian compression formed or "squeezed out" Ordovician-Silurian synclines; they consider Variscan deformation to be of platform-type. Laramide and Late Alpine platform-type faults also deformed the Holy Cross Mts. Ordovician haematites show 3 generations of folds in the Brzeziny Syncline, showing it to be over 250 m in amplitude. This structure is thus not a mesostructure but a large-scale structure formed as a result of orogenic compression. The Devonian-Carboniferous cover shows a platform tectonic style. Differences in style between the folded Cambro-Silurian basement and the unconformably overlying Devonian-Carboniferous sedimentary cover are great and cannot be explained in terms of different rock competence. These tectonic relationships are supported by borehole and geophysical evidence. The Caledonian faulting style is identical in the southern part of the Holy Cross Mts. and the northern Łysogóry area. Laramide and Late Alpine stresses are likely related to Atlantic ocean-floor spreading; stresses acting on the crystalline margin of the East European Craton rejuvenated tectonic lineaments in the Holy Cross Mts. and deformed the Devonian-Cenozoic cover throughout the Polish Lowlands