Dynamics inherent in helix 27 from Escherichia coli 16S ribosomal RNA

The original interpretation of a series of genetic studies suggested that the highly conserved Escherichia coli 16S ribosomal RNA helix 27 (H27) adopts two alternative secondary structure motifs, the 885 and 888 conformations, during each cycle of amino acid incorporation. Recent crystallographic and genetic evidence has called this hypothesis into question. To ask whether a slippery sequence such as that of H27 may harbor inherent conformational dynamics, we have designed a series of model RNAs based on E. coli H27 for in vitro physicochemical studies. One-dimensional (1)H NMR spectroscopy demonstrates that both the 885 and 888 conformations are occupied to approximately the same extent (f(888) = 0.427 +/- 0.04) in the native H27 sequence at low pH (6.4) and low ionic strength (50 mM NaCl). UV irradiation assays conducted under conditions analogous to those used for assays of ribosomal function (pH 7.5 and 20 mM MgCl(2)) suggest that nucleotides 892 and 905, which are too far apart in the known 885 crystal structures, can approach each other closely enough to form an efficient cross-link. The use of a fluorescence resonance energy transfer (FRET)-labeled RNA together with a partially complementary DNA oligonucleotide that induces a shift to the 888 conformation shows that H27 interchanges between the 885 and 888 conformations on the millisecond time scale, with an equilibrium constant of 0.33 +/-0.12. FRET assays also show that tetracycline interferes with the induced shift to the 888 conformation, a finding that is consistent with crystallographic localization of tetracycline bound to the 885 conformation of H27 in the 30S ribosomal subunit. Taken together, our data demonstrate the innate tendency of an isolated H27 to exist in a dynamic equilibrium between the 885 and 888 conformations. This begs the question of how these inherent structural dynamics are suppressed within the context of the ribosome.     

Premature myocardial infarction novel susceptibility locus on chromosome 1P34-36 identified by genomewide linkage analysis

The most frequent causes of death and disability in the Western world are atherosclerotic coronary artery disease (CAD) and acute myocardial infarction (MI). This common disease is thought to have a polygenic basis with a complex interaction with environmental factors. Here, we report results of a genomewide search for susceptibility genes for MI in a well-characterized U.S. cohort consisting of 1,613 individuals in 428 multiplex families with familial premature CAD and MI: 712 with MI, 974 with CAD, and average age of onset of 44.4+/-9.7 years. Genotyping was performed at the National Heart, Lung, and Blood Institute Mammalian Genotyping Facility through use of 408 markers that span the entire human genome every 10 cM. Linkage analysis was performed with the modified Haseman-Elston regression model through use of the SIBPAL program. Three genomewide scans were conducted: single-point, multipoint, and multipoint performed on of white pedigrees only (92% of the cohort). One novel significant susceptibility locus was detected for MI on chromosomal region 1p34-36, with a multipoint allele-sharing P value of <10(-12) (LOD=11.68). Validation by use of a permutation test yielded a pointwise empirical P value of.00011 at this locus, which corresponds to a genomewide significance of P<.05. For the less restrictive phenotype of CAD, no genetic locus was detected, suggesting that CAD and MI may not share all susceptibility genes. The present study thus identifies a novel genetic-susceptibility locus for MI and provides a framework for the ultimate cloning of a gene for the complex disease MI.     

Identification and functional characterization of an N-terminal oligomerization domain for polycystin-2

Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited cause of kidney failure, is caused by mutations in either PKD1 (85%) or PKD2 (15%). The PKD2 protein, polycystin-2 (PC2 or TRPP2), is a member of the transient receptor potential (TRP) superfamily and functions as a non-selective calcium channel. PC2 has been found to form oligomers in native tissues suggesting that it may form functional homo- or heterotetramers with other subunits, similar to other TRP channels. Our experiments unexpectedly revealed that PC2 mutant proteins lacking the known C-terminal dimerization domain were still able to form oligomers and co-immunoprecipitate full-length PC2, implying the possible existence of a proximal dimerization domain. Using yeast two-hybrid and biochemical assays, we have mapped an alternative dimerization domain to the N terminus of PC2 (NT2-1-223, L224X). Functional characterization of this domain demonstrated that it was sufficient to induce cyst formation in zebrafish embryos and inhibit PC2 surface currents in mIMCD3 cells probably by a dominant-negative mechanism. In summary, we propose a model for PC2 assembly as a functional tetramer which depends on both C- and N-terminal dimerization domains. These results have significant implications for our understanding of PC2 function and disease pathogenesis in ADPKD and provide a new strategy for studying PC2 function.     

Real-Time PCR Detection of Brucella abortus: a Comparative Study of SYBR Green I, 5′-Exonuclease, and Hybridization Probe Assays

Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5′-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.     

A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture.

The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pairing between the U2 small nuclear (sn)RNA and a complementary consensus sequence of the intron, called the branch site, results in positioning of the 2'OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes representing the paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine (psi) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide on the intron strand, one of which forms the branch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of psi at the conserved position, however, was accompanied by marked deviation from helical parameters and an extrahelical orientation for the unpaired adenosine. Incorporation of psi also stabilized the branch-site interaction, contributing -0.7 kcal/mol to duplex deltaG degrees 37. These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and imply that the extrahelical orientation of the branch-site adenosine may facilitate recognition of this base during splicing.     

Metastable behavior in Markov processes with internal states

A perturbation framework is developed to analyze metastable behavior in stochastic processes with random internal and external states. The process is assumed to be under weak noise conditions, and the case where the deterministic limit is bistable is considered. A general analytical approximation is derived for the stationary probability density and the mean switching time between metastable states, which includes the pre exponential factor. The results are illustrated with a model of gene expression that displays bistable switching. In this model, the external state represents the number of protein molecules produced by a hypothetical gene. Once produced, a protein is eventually degraded. The internal state represents the activated or unactivated state of the gene; in the activated state the gene produces protein more rapidly than the unactivated state. The gene is activated by a dimer of the protein it produces so that the activation rate depends on the current protein level. This is a well studied model, and several model reductions and diffusion approximation methods are available to analyze its behavior. However, it is unclear if these methods accurately approximate long-time metastable behavior (i.e., mean switching time between metastable states of the bistable system). Diffusion approximations are generally known to fail in this regard.     

三种绿叶挥发性物质对云南切梢小蠹寄主定位行为的干扰作用

为了研究环境中非寄主阔叶植物释放出的绿叶挥发性物质（GLVs）对针叶树蛀干害虫云南切梢小蠹Tomicus yunnanesis的影响, 选取了(E)-2-己烯醛、 (E)-2-己烯醇和(Z)-3-己烯醇3种释放量较大的绿叶挥发性物质, 通过室内松梢取食试验测试了单组分及两两混合后对云南切梢小蠹寄主定位行为的干扰作用。结果表明： 源于阔叶植物的3种绿叶挥发性物质及其混合物能够不同程度干扰云南切梢小蠹的寄主定位行为。当虫放入广口瓶12 h后, 3个单组分绿叶挥发性物质处理组［A： (E)-2-己烯醛, P＜0.01; B： (E)-2-己烯醇, P＜0.01; C： (Z)-3-己烯醇, P＜0.01］及2个混合组分［D： (E)-2-己烯醛+(E)-2-己烯醇, P＜0.01）; E： (E)-2-己烯醛+(Z)-3-己烯醇, P＜0.01］中滞留在松梢外部的虫数与对照组相比都有显著性差异, 绿叶挥发性物质的存在显著降低了云南切梢小蠹侵害云南松松梢的概率。但是, 24 h后只有D组（P＜0.01）和E组（P＜0.01）滞留在松梢外部的虫数与对照组相比具有显著性差异, 在48 h后只有D组（P＜0.01）与对照相比仍具有显著性差异。本研究为利用非寄主植物的次生代谢产物防治云南切梢小蠹进行了有益的探索。     

Solution structure of an alternate conformation of helix27 from Escherichia coli16S rRNA

Helix (H)27 of 16S ribosomal (r)RNA from Escherichia coli was dubbed the "switch helix" when mutagenesis suggested that two alternative base pair registers may have distinct functional roles in the bacterial ribosome. Although more recent genetic analyses suggest that H27 conformational switching is not required for translation, previous solution studies demonstrated that the isolated E. coli H27 can dynamically convert between the 885 and 888 conformations. Here, we have solved the nuclear magnetic resonance solution structure of a locked 888 conformation. NOE and residual dipolar coupling restraints reveal an architecture that markedly differs from that of the 885 conformation found in crystal structures of the bacterial ribosome. In place of the loop E motif that characterizes the 885 conformer and that the 888 conformer cannot adopt, we find evidence for an asymmetrical A-rich internal loop stabilized by stacking interactions among the unpaired A's. Comparison of the isolated H27 888 solution structure with the 885 crystal structure within the context of the ribosome suggests a difference in overall length of H27 that presents one plausible reason for the absence of H27 conformational switching within the sterically confining ribosome.