Stepwise sRNA targeting of structured bacterial mRNAs leads to abortive annealing

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The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA

Small noncoding RNAs (sRNAs) regulate the response of bacteria to environmental stress in conjunction with the Sm-like RNA binding protein Hfq. DsrA sRNA stimulates translation of the RpoS stress response factor in Escherichia coli by base-pairing with the 5′ leader of the rpoS mRNA and opening a stem–loop that represses translation initiation. We report that rpoS leader sequences upstream of this stem–loop greatly increase the sensitivity of rpoS mRNA to Hfq and DsrA. Native gel mobility shift assays show that Hfq increases the rate of DsrA binding to the full 576 nt rpoS leader as much as 50-fold. By contrast, base-pairing with a 138-nt RNA containing just the repressor stem–loop is accelerated only twofold. Deletion and mutagenesis experiments showed that sensitivity to Hfq requires an upstream AAYAA sequence. Leaders long enough to contain this sequence bind Hfq tightly and form stable ternary complexes with Hfq and DsrA. A model is proposed in which Hfq recruits DsrA to the rpoS mRNA by binding both RNAs, releasing the self-repressing structure in the mRNA. Once base-pairing between DsrA and rpoS mRNA is established, interactions between Hfq and the mRNA may stabilize the RNA complex by removing Hfq from the sRNA.     

Lack of Association of the N-acetyltransferase NAT1*10 Allele with Prostate Cancer Incidence, Grade, or Stage Among Smokers in Finland

Genetic variations in xenobiotic metabolizing genes can influence susceptibility to many environmentally induced cancers. Inheritance of the N-acetyltransferase 1 allele (NAT1*10), linked with increased metabolic activation of pro-carcinogens, is associated with an increased susceptibility to many cancers in which cigarette- or meat-derived carcinogens have been implicated in their etiology. The role of NAT1*10 in prostate cancer is under studied. Although cigarette smoking is not considered a risk factor for prostate cancer, a recent review suggests it may play a role in disease progression. Consequently, we examined the association of NAT1*10 with prostate cancer risk, grade, and stage among 400 Finnish male smokers using a case-control study design. Following genotyping of 206 patients and 196 healthy controls, our results do not support the role of NAT1*10 in relation to prostate cancer risk (OR?=?1.28; 95% CI, 0.66-2.47), aggressive disease (OR?=?0.58; 95% CI, 0.13-2.67), or advanced disease (OR?=?1.19; 95% CI, 0.49-2.91).     

Facilitation of group I splicing in vivo: misfolding of the Tetrahymena IVS and the role of ribosomal RNA exons.

Self-splicing of the group I IVS from Tetrahymena thermophila rDNA is limited by the time required for the RNA to reach its active conformation. In vitro, folding is slow because the pre-rRNA becomes kinetically trapped in inactive structures. In vivo, splicing is 50 times more rapid, implying that misfolding of the pre-rRNA is corrected. Exon mutations that inhibit self-splicing 100-fold in vitro were fully rescued when the pre-rRNA containing the IVS was expressed in E. coli. In contrast, IVS mutations that cause misfolding were only partially suppressed at 42 degrees C, and doubled the activation energy of splicing. These results suggest that intracellular folding of the pre-rRNA involves metastable intermediates similar to those observed in vitro. Precursors with natural rRNA exons were more active and less cold-sensitive than those with non-rRNA exons. This shows that the rRNA reduces misfolding of the IVS, thereby facilitating splicing of the pre-rRNA in vivo.     

Saturation dependency of the Bohr effect: interactions among H-+, CO2, and DPG.

The Bohr effect was measured in normal whole blood and in blood with low DPG concentration as a function of oxygen saturation. pH was changed by varying CO2 concentration (CO2 Bohr effect) or by addition of isotonic NaOH or HC1 at constant PCO2 (fixed acid Bohr effect). At nornal DPG concentration CO2 Bohr effect was -0.52 at 50% blood oxygen saturation, increasing in magnitude at lower saturation and decreasing in magnitude at higher saturation. In DPG depleted blood with base excess (BE) similar to 0 meq/1, there was similar dependence of CO2 Bohr effect on oxygen saturation. At BE similar to -10 meq/1, influence of saturation was comparable, but the magnitude of the Bohr effect was markedly increased at all saturations. Fixed acid Bohr effect at normal DPG concentration was -0.45 at saturations of 50-90% but decreased at lower saturations. In DPG-depleted blood fixed acid Bohr effect averaged about -0.33 with minimal variation with saturation. Influence of DPG on oxygen affinity was greater at intermediate saturations and less at saturations below 20% and above 80%. Effect of CO2, independent of pH, was many fold greater at lower oxygen saturations than at higher saturations. These results support the suggestion that the alpha chain of hemoglobin is the site of the initial oxygenation reaction. Physiologically they indicate that the relative contribution of CO2 and fixed acid, as well as the level of oxygen saturation and DPG concentration, may be important in determining PO2 of capillary blood and resulting oxygen delivery.