Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1.

Tight binding of Gdown1 represses RNA polymerase II (Pol II) function in a manner that is reversed by Mediator, but the structural basis of these processes is unclear. Although Gdown1 is intrinsically disordered, its Pol II interacting domains were localized and shown to occlude transcription factor IIF (TFIIF) and transcription factor IIB (TFIIB) binding by perfect positioning on their Pol II interaction sites. Robust binding of Gdown1 to Pol II is established by cooperative interactions of a strong Pol II binding region and two weaker binding modulatory regions, thus providing a mechanism both for tight Pol II binding and transcription inhibition and for its reversal. In support of a physiological function for Gdown1 in transcription repression, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei of early Drosophila embryos but re-localizes to the cytoplasm during zygotic genome activation. Our study reveals a self-inactivation through Gdown1 binding as a unique mode of repression in Pol II function

How the DNA sequence affects the Hill curve of transcriptional response

The Hill coefficient is often used as a direct measure of the cooperativity of binding processes. It is an essential tool for probing properties of reactions in many biochemical systems. Here we analyze existing experimental data and demonstrate that the Hill coefficient characterizing the binding of transcription factors to their cognate sites can in fact be larger than one -- the standard indication of cooperativity -- even in the absence of any standard cooperative binding mechanism. By studying the problem analytically, we demonstrate that this effect occurs due to the disordered binding energy of the transcription factor to the DNA molecule and the steric interactions between the different copies of the transcription factor. We show that the enhanced Hill coefficient implies a significant reduction in the number of copies of the transcription factors which is needed to occupy a cognate site and, in many cases, can explain existing estimates for numbers of the transcription factors in cells. The mechanism is general and should be applicable to other biological recognition processes.Comment: 9 pages, 7 figure

SGLT5 Reabsorbs Fructose in the Kidney but Its Deficiency Paradoxically Exacerbates Hepatic Steatosis Induced by Fructose

Although excessive fructose intake is epidemiologically linked with dyslipidemia, obesity, and diabetes, the mechanisms regulating plasma fructose are not well known. Cells transfected with sodium/glucose cotransporter 5 (SGLT5), which is expressed exclusively in the kidney, transport fructose in vitro; however, the physiological role of this transporter in fructose metabolism remains unclear. To determine whether SGLT5 functions as a fructose transporter in vivo, we established a line of mice lacking the gene encoding SGLT5. Sodium-dependent fructose uptake disappeared in renal brush border membrane vesicles from SGLT5-deficient mice, and the increased urinary fructose in SGLT5-deficient mice indicated that SGLT5 was the major fructose reabsorption transporter in the kidney. From this, we hypothesized that urinary fructose excretion induced by SGLT5 deficiency would ameliorate fructose-induced hepatic steatosis. To test this hypothesis we compared SGLT5-deficient mice with wild-type mice under conditions of long-term fructose consumption. Paradoxically, however, fructose-induced hepatic steatosis was exacerbated in the SGLT5-deficient mice, and the massive urinary fructose excretion was accompanied by reduced levels of plasma triglycerides and epididymal fat but fasting hyperinsulinemia compared with fructose-fed wild-type mice. There was no difference in food consumption, water intake, or plasma fructose between the two types of mice. No compensatory effect by other transporters reportedly involved in fructose uptake in the liver and kidney were indicated at the mRNA level. These surprising findings indicated a previously unrecognized link through SGLT5 between renal fructose reabsorption and hepatic lipid metabolism

Acceleration of Age-Related Changes in the Retina in ␣-Tocopherol Transfer Protein Null Mice Fed a Vitamin E-Deficient Diet

PURPOSE. To assess the role of vitamin E (VE) in age-related changes in the retinal tissues by using a mouse model of severe VE deficiency. METHODS. Pups of ␣-tocopherol transfer protein null (a-TTP Ϫ/Ϫ ) mice were fed a VE-deficient diet for 4 or 18 months (VE (Ϫ) group). Wild-type C57BL/6 mice were fed a 0.002% ␣-tocopherol-supplemented diet (VE (ϩ) group). In various ocular tissues, the VE levels were measured by high-performance liquid chromatography; the fatty acid composition by gas chromatography (GC); and the hydroxyoctadecadienoic acid and 8-iso-prostaglandin F 2␣ levels, which are biomarkers for lipid peroxidation, by GC-mass spectrometry. The retinal structure was assessed by light, electron, and fluorescence microscopy. RESULTS. The ␣-tocopherol level in the retinas obtained from 4-month-old VE (Ϫ) animals was 71-fold lower than that in the retinas obtained from the VE (ϩ) group. In addition, ␥-tocopherol was not detected; thus, the VE (Ϫ) group demonstrated a more severe VE deficiency than ever reported. In this group, the concentration of n-3 polyunsaturated fatty acids decreased (0.3-to 0.9-fold), whereas that of other classes of fatty acids was unchanged or increased. At 18 months of age, the number of the outer nuclear layer (ONL) nuclei was observed to be 17% lower in the VE (Ϫ) than in the VE (ϩ) group (P Ͻ 0.05). Electron microscopy revealed larger amounts of matrix between the ONL nuclei indicating the Müller cell hypertrophy, greatly expanded rod outer segment discs, and a larger number of inclusion bodies in the retinal pigment epithelium (RPE; P Ͻ 0.05) in the VE (Ϫ) group. Fluorescence microscopy revealed that the autofluorescence signal was increased in the RPE layer in this group. When the observations of the 18-month-old animals were compared to those of the 4-month-old animals, the hydroxyoctadecadienoic acid and 8-iso-prostaglandin F 2␣ levels were found to be increased in the retina and RPE obtained from both the VE (Ϫ) and VE (ϩ) groups; however, the age-related increases were more remarkable in the VE (Ϫ) group (2.6-to 43.5-fold) than in the VE (ϩ) group (0.8-to 8.7-fold). CONCLUSIONS. The combined use of a-TTP Ϫ/Ϫ mice and a VEdeficient diet leads to a severe deficiency of VE, enhances lipid peroxidation in the retina, and accelerates degenerative damage of the retina with age. (Invest Ophthalmol Vis Sci. 2007; 48:396 -404

Mutations in two global regulators lower individual mortality in Escherichia coli

There has been considerable investigation into the survival of bacterial cells under stress conditions, but little is known about the causes of mortality in the absence of exogenous stress. That there is a basal frequency of cell death in such populations may reflect that it is either impossible to avoid all lethal events, or alternatively, that it is too costly. Here, through a genetic screen in the model organism Escherichia coli, we identify two mutants with lower frequencies of mortality: rssB and fliA. Intriguingly, these two genes both affect the levels of different sigma factors within the cell. The rssB mutant displays enhanced resistance to multiple external stresses, possibly indicating that the cell gains its increased vitality through elevated resistance to spontaneous, endogenous stresses. The loss of fliA does not result in elevated stress resistance; rather, its survival is apparently due to a decreased physical stress linked to the insertion of the flagellum through the membrane and energy saved through the loss of the motor proteins. The identification of these two mutants implies that reducing mortality is not impossible; rather, due to its cost, it is subject to trade-offs with other traits that contribute to the competitive success of the organism

The E. coli Anti-Sigma Factor Rsd: Studies on the Specificity and Regulation of Its Expression

Background: Among the seven different sigma factors in E. coli s 70 has the highest concentration and affinity for the core RNA polymerase. The E. coli protein Rsd is regarded as an anti-sigma factor, inhibiting s 70-dependent transcription at the onset of stationary growth. Although binding of Rsd to s 70 has been shown and numerous structural studies on Rsd have been performed the detailed mechanism of action is still unknown. Methodology/Principal Findings: We have performed studies to unravel the function and regulation of Rsd expression in vitro and in vivo. Cross-linking and affinity binding revealed that Rsd is able to interact with s 70, with the core enzyme of RNA polymerase and is able to form dimers in solution. Unexpectedly, we find that Rsd does also interact with s 38, the stationary phase-specific sigma factor. This interaction was further corroborated by gel retardation and footprinting studies with different promoter fragments and s 38-ors 70-containing RNA polymerase in presence of Rsd. Under competitive in vitro transcription conditions, in presence of both sigma factors, a selective inhibition of s 70-dependent transcription was prevailing, however. Analysis of rsd expression revealed that the nucleoid-associated proteins H-NS and FIS, StpA and LRP bind to the regulatory region of the rsd promoters. Furthermore, the major promoter P2 was shown to be down-regulated in vivo by RpoS, the stationary phase-specific sigma factor and the transcription factor DksA, while induction of the stringent control enhanced rsd promoter activity. Most notably, the dam-dependent methylation of a cluster of GATC sites turned ou

Promoter and regulon analysis of nitrogen assimilation factor, σ54, reveal alternative strategy for E. coli MG1655 flagellar biosynthesis

Bacteria core RNA polymerase (RNAP) must associate with a σ factor to recognize promoter sequences. Promoters recognized by the σ54 (or σN) associated RNA polymerase are unique in having conserved positions around −24 and −12 nucleotides upstream from the transcriptional start site. Using DNA microarrays representing the entire Escherichia coli genome and promoter validation approaches, we identify 40 in vivo targets of σ54, the nitrogen assimilation σ factor, and estimate that there are 70 σ54 promoters in total. Immunoprecipitation assays have been performed to further evaluate the efficiency of our approaches. In addition, promoter consensus binding search and primer extension assay helped us to identify a new σ54 promoter carried by insB-5 in the upstream of flhDC operon. The involvement of σ54 in flagellar biosynthesis in sequenced E. coli strain MG1655 indicates a fluid gene regulation phenomenon carried by some mobile elements in bacteria genome

Insights into the complex regulation of rpoS in Borrelia burgdorferi

Co-ordinated regulation of gene expression is required for the transmission and survival of Borrelia burgdorferi in different hosts. The sigma factor RpoS (σS), as regulated by RpoN (σ54), has been shown to regulate key virulence factors (e.g. OspC) required for these processes. As important, multiple signals (e.g. temperature, pH, cell density, oxygen) have been shown to increase the expression of σS-dependent genes; however, little is known about the signal transduction mechanisms that modulate the expression of rpoS. In this report we show that: (i) rpoS has a σ54-dependent promoter that requires Rrp2 to activate transcription; (ii) Rrp2Δ123, a constitutively active form of Rrp2, activated σ54-dependent transcription of rpoS/P-lacZ reporter constructs in Escherichia coli; (iii) quantitative reverse transcription polymerase chain reaction (QRT-PCR) experiments with reporter cat constructs in B. burgdorferi indicated that Rrp2 activated transcription of rpoS in an enhancer-independent fashion; and finally, (iv) rpoN is required for cell density- and temperature-dependent expression of rpoS in B. burgdorferi, but histidine kinase Hk2, encoded by the gene immediately upstream of rrp2, is not essential. Based on these findings, a model for regulation of rpoS has been proposed which provides mechanisms for multiple signalling pathways to modulate the expression of the σS regulon in B. burgdorferi

6S RNA regulation of relA alters ppGpp levels in early stationary phase

6S RNA is a small, non-coding RNA that interacts directly with σ70-RNA polymerase and regulates transcription at many σ70-dependent promoters. Here, we demonstrate that 6S RNA regulates transcription of relA, which encodes a ppGpp synthase. The 6S RNA-dependent regulation of relA expression results in increased ppGpp levels during early stationary phase in cells lacking 6S RNA. These changes in ppGpp levels, although modest, are sufficient to result in altered regulation of transcription from σ70-dependent promoters sensitive to ppGpp, including those promoting expression of genes involved in amino acid biosynthesis and rRNA. These data place 6S RNA as another player in maintaining appropriate gene expression as cells transition into stationary phase. Independent of this ppGpp-mediated 6S RNA-dependent regulation, we also demonstrate that in later stationary phase, 6S RNA continues to downregulate transcription in general, and specifically at a subset of the amino acid promoters, but through a mechanism that is independent of ppGpp and which we hypothesize is through direct regulation. In addition, 6S RNA-dependent regulation of σS activity is not mediated through observed changes in ppGpp levels. We suggest a role for 6S RNA in modulating transcription of several global regulators directly, including relA, to downregulate expression of key pathways in response to changing environmental conditions

The metabolic cost of flagellar motion in Pseudomonas putida KT2440

Although the flagellar machinery of environmental bacteria endows cells with a phenomenal survival device, it also consumes much of the metabolic currency necessary for fuelling such a vigorous nanomotor. The physiological cost of flagella-related functions of the soil bacterium Pseudomonas putida KT2440 was examined and quantified through the deletion of a ∼ 70 kb DNA segment of the genome (∼ 1.1%), which includes relevant structural and regulatory genes in this micro-organism. The resulting strain lacked the protruding polar cords that define flagella in the wild-type P. putida strain and was unable of any swimming motility while showing a significant change in surface hydrophobicity. However, these deficiencies were otherwise concomitant with clear physiological advantages: rapid adaptation of the deleted strain to both glycolytic and gluconeogenic carbon sources, increased energy charge and, most remarkably, improved tolerance to oxidative stress, reflecting an increased NADPH/NADP+ ratio. These qualities improve the endurance of nonflagellated cells to the metabolic fatigue associated with rapid growth in rich medium. Thus, flagellar motility represents the archetypal tradeoff involved in acquiring environmental advantages at the cost of a considerable metabolic burden.This study was supported by the BIO and FEDER CONSOLIDER-INGENIO Program, the MICROME, STFLOW and ARISYS Contracts of the EU, the ERANET-IB program and the PROMT Project of the CAM.Peer reviewe