RNA tertiary interactions mediate native collapse of a bacterial group I ribozyme

Large RNAs collapse into compact intermediates in the presence of counterions before folding to the native state. We previously found that collapse of a bacterial group I ribozyme correlates with the formation of helices within the ribozyme core, but occurs at Mg2+ concentrations too low to support stable tertiary structure and catalytic activity. Here, using small-angle X-ray scattering, we show that Mg2+-induced collapse is a cooperative folding transition that can be fit by a two-state model. The Mg2+ dependence of collapse is similar to the Mg2+ dependence of helix assembly measured by partial ribonuclease T1 digestion and of an unfolding transition measured by UV hypochromicity. The correspondence between multiple probes of RNA structure further supports a two-state model. A mutation that disrupts tertiary contacts between the L9 tetraloop and its helical receptor destabilized the compact state by 0.8 kcal/mol, while mutations in the central triplex were less destabilizing. These results show that native tertiary interactions stabilize the compact folding intermediates under conditions in which the RNA backbone remains accessible to solvent.     

ANTIPYRETIC ACTION OF DEXAMETHASONE ON EGTAZIC ACIDINDUCED FEVER IN RABBITS

本文用脑室灌注和Ｆｕｒａ２测定细胞内游离钙技术观察了地塞米松（ｄｅｘａｍｅｔｈａｓｏｎｅ,ＤＥＸ）对家兔乙二醇双（２氨基乙醚）四乙酸性发热效应和下丘脑细胞内游离钙浓度（［Ｃａ２＋］ｉ）的影响,借此深入探讨地塞米松解热作用的中枢机制。结果发现：脑室灌注乙二醇双（２氨基乙醚）四乙酸（０６ｎｍｏｌ）引起家兔结肠温度明显升高,静脉注射地塞米松（５ｍｇ／ｋｇ）显著抑制家兔乙二醇双（２氨基乙醚）四乙酸性发热,地塞米松（６０～１２０μｍｏｌ／Ｌ）并不影响下丘脑细胞内［Ｃａ２＋］ｉ,而事先脑室灌注抑制基因转录的放线菌素Ｄ（３ｎｍｏｌ）则完全取消了地塞米松对乙二醇双（２氨基乙醚）四乙酸性发热的解热作用。这些结果提示：地塞米松显著抑制家兔乙二醇双（２氨基乙醚）四乙酸性发热,其机制与地塞米松激活脑内某些基因的表达有关,而与下丘脑神经细胞跨膜钙离子流无关。     

Compaction of a bacterial group I ribozyme coincides with the assembly of core helices

Counterions are critical to the self-assembly of RNA tertiary structure because they neutralize the large electrostatic forces which oppose the folding process. Changes in the size and shape of the Azoarcus group I ribozyme as a function of Mg(2+) and Na(+) concentration were followed by small angle neutron scattering. In low salt buffer, the RNA was expanded, with an average radius of gyration (R(g)) of 53 +/- 1 A. A highly cooperative transition to a compact form (R(g) = 31.5 +/- 0.5 A) was observed between 1.6 and 1.7 mM MgCl(2). The collapse transition, which is unusually sharp in Mg(2+), has the characteristics of a first-order phase transition. Partial digestion with ribonuclease T1 under identical conditions showed that this transition correlated with the assembly of double helices in the ribozyme core. Fivefold higher Mg(2+) concentrations were required for self-splicing, indicating that compaction occurs before native tertiary interactions are fully stabilized. No further decrease in R(g) was observed between 1.7 and 20 mM MgCl(2), indicating that the intermediates have the same dimensions as the native ribozyme, within the uncertainty of the data (+/-1 A). A more gradual transition to a final R(g) of approximately 33.5 A was observed between 0.45 and 2 M NaCl. This confirms the expectation that monovalent ions not only are less efficient in charge neutralization but also contract the RNA less efficiently than multivalent ions.     

Noninvasive Neutron Scattering Measurements Reveal Slower Cholesterol Transport in Model Lipid Membranes

Proper cholesterol transport is essential to healthy cellular activity and any abnormality can lead to several fatal diseases. However, complete understandings of cholesterol homeostasis in the cell remains elusive, partly due to the wide variability in reported values for intra- and intermembrane cholesterol transport rates. Here, we used time-resolved small-angle neutron scattering to measure cholesterol intermembrane exchange and intramembrane flipping rates, in situ, without recourse to any external fields or compounds. We found significantly slower transport kinetics than reported by previous studies, particularly for intramembrane flipping where our measured rates are several orders of magnitude slower. We unambiguously demonstrate that the presence of chemical tags and extraneous compounds employed in traditional kinetic measurements dramatically affect the system thermodynamics, accelerating cholesterol transport rates by an order of magnitude. To our knowledge, this work provides new insights into cholesterol transport process disorders, and challenges many of the underlying assumptions used in most cholesterol transport studies to date.