Base-pair probability profiles of RNA secondary structures

Dynamic programming algorithms are able to predict optimal andsuboptimal secondary structures of RNA. These suboptimal oralternative secondary structures are important for the biologicalfunction of RNA. The distribution of secondary structures presentin solution is governed by the thermodynamic equilibrium betweenthe different structures. An algorithm is presented which approximatesthe total partition function by a Boltzmann–weighted summationof optimal and suboptimal secondary structures at several temperatures.A clear representation of the equilibrium distribution of secondarystructures is derived from a two-dimensional bonding matrixwith base–pairing probability as the third dimension.The temperature dependence of the equilibrium distribution givesthe denaturation behavior of the nucleic acid, which may becompared to experimental optical denaturation curves after correctionfor the hypochromicities of the different base-pairs. Similarly,temperature-induced mobility changes detected in temperature-gradientgel electrophoresis of nucleic acids may be interpreted on thebasis of the temperature dependence of the equilibrium distribution.Results are illustrated for natural circular and synthetic linearpotato spindle tuber viroid RNA respectively, and are comparedto experimental data.     

Deoxyribose conformation in [d(GTATATAC)]2: evaluation of sugar pucker by simulation of double-quantum-filtered COSY cross-peaks

Exchangeable and nonexchangeable proton and phosphorus resonances (11.75 T) of [d(GTATATAC)]2 in aqueous solution were assigned by using proton two-dimensional nuclear Overhauser effect (2D NOE) spectra, homonuclear proton double-quantum-filtered COSY (2QF-COSY) spectra, proton spin-lattice relaxation time measurements, and 31P1H heteronuclear shift correlation spectra. Due to the large line widths, it was not possible to directly extract vicinal proton coupling constant values from any spectrum including ECOSY or 2QF-COSY. However, comparison of quantitative 2QF-COSY spectral simulations with experimental spectra enabled elucidation of coupling constants. The scope and limitations of this approach were explored by computation and by use of experimental data. It was found that proton line widths exhibit some variability from one residue to the next as well as from one proton to the next within a residue and the exact line width is critical to accurate evaluation of coupling constants. Experimental 2QF-COSY spectra were not consistent with a rigid deoxyribose conformation for any of the nucleotide residues. A classical two-state model, with rapid jumps between C2'-endo (pseudorotation angle P = 162 degrees) and C3'-endo (P = 9 degrees) conformations, was able to account for the spectral characteristics of terminal residue sugars: 60% C2'-endo and 40% C3'-endo. However, the 2QF-COSY cross-peaks from the -TATATA- core could be simulated only if the classical two-state model was altered such that the dominant conformer had a pseudorotation angle at 144 degrees instead of 162 degrees. In this case, the major conformer amounted to 80-85%. Alternatively, the spectral data were consistent with a three-state model in which C2'-endo and C3'-endo conformations had the largest and smallest populations, respectively, but a third conformer corresponding to C1'-exo (P = 126 degrees) was present, consistent with recent molecular dynamics calculations. This alternative yielded populations of 50% (P = 162 degrees), 35% (P = 126 degrees), and 15% (P = 9 degrees) for the -TATATA- sugars. The spectral results indicate little variation of sugar pucker between T and A. Small differences in cross-peak component intensities and characteristic spectral distortions, however, do suggest some unquantified variation. 31P1H heteronuclear chemical shift correlation spectra manifested alternating chemical shifts and coupling constants suggestive of phosphodiester backbone conformational differences between TA and AT junctions.     

Production of 3-acetoxyscirpene-4,15-diol from anguidine (4,15-diacetoxyscirpene-3-ol) by Fusarium oxysporum f.sp. vasinfectum.

Growing cells of Fusarium oxysporum f.sp. vasinfectum (ATCC 7808) formed 3-acetoxyscirpene-4,15-diol from anguidine (4,15-diacetoxyscirpene-3-ol) by way of the intermediates triacetoxyscirpene, 3,4-diacetoxyscirpene-15-ol and 3,15-diacetoxyscirpene-4-ol. The new 3-acetoxy analog was found to be less active than anguidine and the other monoacetoxy derivatives when tested against a series of fungal strains and against HeLa cells in vitro.     

The interaction of RNA polymerase and lac repressor with the lac control region.

We have examined the interactions of lac repressor and RNA polymerase with the DNA of the lac control region, using a method for direct visualization of the regions of DNA protected by proteins from DNAase attack. The repressor protects the operator essentially as reported by Gilbert and Maxam (1) with some small modifications. However, the evidence reported here concerning the binding of RNA polymerase to the DNA of the promoter mutant UV5 indicates that : 1) the RNA polymerase molecule binds asymmetrically to the promoter DNA, 2) RNA polymerase protects DNA sequences to within a few bases of the CAP binding site, suggesting direct interaction between polymerase and the CAP protein at this site, 3) RNA polymerase still binds to the promoter when repressor is bound to the operator, but fails to form the same extensive complex.     

Hypothetical Outcome Plots Outperform Error Bars and Violin Plots for Inferences about Reliability of Variable Ordering

Many visual depictions of probability distributions, such as error bars, are difficult for users to accurately interpret. We present and study an alternative representation, Hypothetical Outcome Plots (HOPs), that animates a finite set of individual draws. In contrast to the statistical background required to interpret many static representations of distributions, HOPs require relatively little background knowledge to interpret. Instead, HOPs enables viewers to infer properties of the distribution using mental processes like counting and integration. We conducted an experiment comparing HOPs to error bars and violin plots. With HOPs, people made much more accurate judgments about plots of two and three quantities. Accuracy was similar with all three representations for most questions about distributions of a single quantity.     

Distinct binding sites of Ala48-hirudin1-47 and Ala48-hirudin48-65 on alpha-thrombin

The interaction of alpha-thrombin with Ala48-hirudin, Ala48-hirudin1-47, and Ala48-hirudin48-65 was analyzed. Mutations at Pro48 were found to cause only slight changes in the kon (human: 3.1 +/- 0.3 x 10(8) M-1 s-1; bovine: 1.03 +/- 0.3 x 10(8) M-1 s-1) and koff (human: 0.4 +/- 0.2 x 10(-3) s-1; bovine: 2.9 +/- 0.4 x 10(-3) s-1) rate constants for the formation of the thrombin-hirudin complex. The amino-terminal fragment Ala48-hirudin1-47 containing the three disulfide bridges and the carboxyl-terminal fragment Ala48-hirudin48-65 were derived from the Ala48 mutant by proteolysis with endoproteinase Lys-C. These fragments inhibit bovine alpha-thrombin clotting activity with IC50 values of 0.6 and 4.9 microM, respectively (2.4 nM for r-hirudin). By mapping the interaction of Ala48-hirudin-derived fragments with bovine alpha-thrombin by limited proteolysis with trypsin and pancreatic elastase distinct binding sites for each fragment were determined. The carboxyl-terminal fragment was found to bind to the proposed anion-binding exosite in the region B62-74, whereas the amino-terminal fragment binds to a region around the elastase cleavage site at residues 150-151 of the alpha-thrombin B-chain.     

The Heat-Resistant Agglutinin Family Includes a Novel Adhesin from Enteroaggregative Escherichia coli Strain 60A

Heat-resistant agglutinin 1 (Hra1) is an accessory colonization factor of enteroaggregative Escherichia coli (EAEC) strain 042. Tia, a close homolog of Hra1, is an invasin and adhesin that has been described in enterotoxigenic E. coli. We devised a PCR-restriction fragment length polymorphism screen for the associated genes and found that they occur among 55 (36.7%) of the enteroaggregative E. coli isolates screened, as well as lower proportions of enterotoxigenic, enteropathogenic, enterohemorrhagic, and commensal E. coli isolates. Overall, 25%, 8%, and 3% of 150 EAEC strains harbored hra1 alone, tia alone, or both genes, respectively. One EAEC isolate, 60A, produced an amplicon with a unique restriction profile, distinct from those of hra1 and tia. We cloned and sequenced the full-length agglutinin gene from strain 60A and have designated it hra2. The hra2 gene was not detected in any of 257 diarrheagenic E. coli isolates in our collection but is present in the genome of Salmonella enterica serovar Heidelberg strain SL476. The cloned hra2 gene from strain 60A, which encodes a predicted amino acid sequence that is 64% identical to that of Hra1 and 68% identical to that of Tia, was sufficient to confer adherence on E. coli K-12. We constructed an hra2 deletion mutant of EAEC strain 60A. The mutant was deficient in adherence but not autoaggregation or invasion, pointing to a functional distinction from the autoagglutinin Hra1 and the Tia invasin. Hra1, Tia, and the novel accessory adhesin Hra2 are members of a family of integral outer membrane proteins that confer different colonization-associated phenotypes.     

Prohibitin is a cholesterol‐sensitive regulator of cell cycle transit

Cholesterol is essential in establishing most functional animal cell membranes; cells cannot grow or proliferate in the absence of sufficient cholesterol. Consequently, almost every cell, tissue, and animal tightly regulates cholesterol homeostasis, including complex mechanisms of synthesis, transport, uptake, and disposition of cholesterol molecules. We hypothesize that cellular recognition of cholesterol insufficiency causes cell cycle arrest in order to avoid a catastrophic failure in membrane synthesis. Here, we demonstrate using unbiased proteomics and standard biochemistry that cholesterol insufficiency causes upregulation of prohibitin, an inhibitor of cell cycle progression, through activation of a cholesterol‐responsive promoter element. We also demonstrate that prohibitin protects cells from apoptosis caused by cholesterol insufficiency. This is the first study tying cholesterol homeostasis to a specific cell cycle regulator that inhibits apoptosis. J. Cell. Biochem. 111: 1367–1374, 2010. © 2010 Wiley‐Liss, Inc.