Transcription. Clamping the TBP stirrup.
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DNA↗
Biomedical subjects
S Hahn
Publications and source records attributed to S Hahn.
Model for binding of transcription factor TFIIB to the TBP-DNA complex.
Transcription factor TFIIB is essential for the formation of RNA polymerase II initiation complexes where it binds to the TATA-binding protein (TBP) complex with DNA and recruits RNA polymerase II. TFIIB is probably a target for various activators. Several models have been proposed for the position of TFIIB in the TFIIB-TBP-DNA complex. Here we examine the structure of this complex using gel mobility-shift assays and hydroxyl-radical footprinting. TFIIB requires at least seven base pairs of DNA on either side of the TATA box to form a stable TFIIB-TBP-DNA complex. The sugar residues protected from hydroxyl-radical cleavage by the TFIIB-TBP complex were mapped on the crystal-structure model of the TBP-DNA complex. This analysis suggests that TFIIB binds beneath the concave surface of TBP, contacting DNA both upstream and downstream of the TATA box. Our model predicts that TFIIB binds close to the C-terminal stirrup of TBP and provides one explanation for why TBP needs to bend DNA.
Carbohydrate Metabolism↗
TFIIIC determines RNA polymerase III specificity at the TATA-containing yeast U6 promoter.
The gene encoding yeast U6 snRNA that is transcribed by RNA polymerase III (Pol III) contains both a TATA box upstream of the transcription start site and a downstream binding site for the factor TFIIIC. This juxtaposition of elements typical of both Pol II- and Pol III-transcribed genes raises the question of how polymerase specificity is determined. The upstream U6 promoter containing the TATA box and transcription start site was shown previously to be transcribed by Pol III in vitro. We therefore tested whether the upstream promoter of yeast U6 encodes Pol III specificity. One model is that polymerase specificity is conferred by the homologous Pol II and Pol III transcription factors TFIIB and BRF1. However, we found no specificity in the binding of BRF1 or TFIIB to TATA-containing promoters of genes specifically transcribed by Pol III or Pol II. Yeast strains deficient for Pol II or Pol III transcription were employed to examine U6 polymerase specificity in vivo. We find that the U6 upstream promoter is Pol II-specific in vivo and is converted to Pol III specificity by TFIIIC. Thus, preferential recruitment of TFIIIB by TFIIIC probably excludes the Pol II general factors and promotes Pol III transcription, thereby determining polymerase specificity.
Base Sequence↗
The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: implications for protection against coronary heart disease.
A number of lines of evidence suggest that red wine may be more effective than other alcoholic beverages in decreasing the risk of coronary heart disease (CHD) mortality. This protection over and above that due to ethanol itself may be explained by phenolic components with which red wines are richly endowed. We have studied the effects of the trihydroxy stilbene trans-resveratrol on human platelet aggregation and on the synthesis of three eicosanoids from arachidonate by platelets, i.e. thromboxane B2 (TxB2), hydroxyheptadecatrienoate (HHT) and 12-hydroxyeicosatetraenoate (12-HETE). These effects were compared with the actions of other wine phenolics (quercetin, catechin and epicatechin) and antioxidants (alpha-tocopherol, hydroquinone and butylated hydroxytoluene). trans-Resveratrol and quercetin demonstrated a dose-dependent inhibition of both thrombin-induced and ADP-induced platelet aggregation, whereas ethanol inhibited only thrombin-induced aggregation. The other compounds tested were inactive. trans-Resveratrol also inhibited the synthesis of TxB2, HHT, and to a lesser extent 12-HETE, from arachidonate in a dose-dependent manner. Quercetin inhibited only 12-HETE synthesis, and hydroquinone caused slight inhibition of TxB2 synthesis, the remaining compounds being ineffective. De-alcoholized red wines inhibited platelet aggregation; their ability to inhibit the synthesis of TxB2 but not that of 12-HETE from labelled arachidonate by washed human platelets was proportional to their trans-resveratrol concentration. These results are consistent with the notion that trans-resveratrol may contribute to the presumed protective role of red wine against atherosclerosis and CHD.
Arteriosclerosis↗
Down-modulation of CD4+ T helper type 2 and type 0 cells by T helper type 1 cells via Fas/Fas-ligand interaction.
Fas was recently demonstrated to be the major target molecule engaged by CD4+ cytolytic T lymphocytes (CTL). We examined Fas expression on various cloned T cell subpopulations and their susceptibility to lysis by CD4+ or CD8+ CTL. A reciprocal relationship in Fas and Fas-ligand expression was observed in CD4+ T helper (Th)1- and Th2-type clones, and Fas mRNA was predominantly detected in Th2 clones, whereas Fas-ligand mRNA was principally found in Th1 clones. The two Th0 clones tested expressed both Fas and Fas-ligand, but only one exhibited cytolytic activity, whereas both were sensitive to CD4-mediated lysis. A functional consequence of the inverse Fas-Fas-ligand expression pattern was that Th2 and Th0 cells were sensitive to lysis by both Th1 CD4+ CTL and a CD8+ CTL clone in a Fas-dependent manner. These results suggest that cytolytic CD4+ Th1 cells may play an immunomodulatory role, regulating a Th2/Th0 response by Fas-mediated lysis.
Animals↗
Psychiatric comorbidity, health status, and functional impairment associated with alcohol abuse and dependence in primary care patients: findings of the PRIME MD-1000 study.
The psychiatric comorbidity, health, and functioning of primary care patients with alcohol abuse and dependence (AAD) were investigated in a sample of 1,000 patients. Psychiatric symptomatology was assessed with the Primary Care Evaluation of Mental Disorders (PRIME-MD) diagnostic system. Health and functional status was assessed with the Medical Outcomes Study Short Form General Health Survey (SF-20). Results indicated that use of the PRIME-MD system brought about a 71% increase in physician recognition of AAD. AAD patients were diagnosed with substantial psychiatric comorbidity, and they reported poorer health and functioning than did patients without any psychiatric disorders. However, they reported less impairment and psychiatric comorbidity than did patients with other psychiatric disorders. Results also indicated that AAD patients' health and functioning were associated with the presence or absence of psychiatric comorbidity.
Adolescent↗
Attentional flexibility and aging: you don't need to be 20 years of age to split the beam.
Whether young and old adults were able to selectively attend to noncontiguous locations in the visual field and ignore physically interspersed distractor stimuli was examined. Participants decided whether 2 letters matched or mismatched. Target letter locations were precued by square boxes on an imaginary circle centered on fixation. Distractors were located between the 2 targets. Young and old were unable to ignore the distractors when the targets and distractors were presented as onset stimuli; however both young and old were able to ignore the distractors when the target and distractors were presented as non-onset stimuli. The time course of attentional allocation was equivalent for young and old. Results are discussed in terms of models of visual selective attention and the flexibility of attentional control.
Adolescent↗
Mechanism and biological significance of CD4-mediated cytotoxicity.
It is now well established that CD4+ T cells can express cytotoxic activity. This type of cell-mediated cytotoxicity is associated with the Th1-, but not with the Th2-phenotype. While the activation of CD4+ CTL is MHC class II-restricted, the effector phase, i.e. the target cell killing is unrestricted and antigen non-specific. In analogy to CD8+ CTL, CD4-mediated target cell death is by DNA fragmentation. However, the molecular mechanism of killing differs from CD8-mediated lysis. Thus, CD4+ CTL preferentially lyse their targets via Fas-Fas ligand interaction, whereas the major cytotoxic effect of CD8+ CTL is by granule exocytosis, i.e. perforin and granzymes. Although CD8+ CTL can also express the FasL, their lytic activity through interaction with Fas is of less importance. Likewise, some CD4+ CTL may also kill by perforin/granzymes activity, but this pathway is of minor significance. The aims of CD8- or CD4-mediated lysis are also different. Thus, the major task of CD8+ CTL which recognize and kill their targets in the context of MHC class I molecules, is the lysis of virally infected cells and battling against tumor cells. CD4+ CTL, on the other hand, have an immunomodulatory role. Thus, they preferentially eliminate activated MHC class II-positive cells, i.e. APC, be they monocytes/macrophages, B cells or T cells. They may lyse these cells in order to prevent an overreaction of the ongoing immune response or in order to remove potentially hazardous cells upon completion of the immune response. The Fas-FasL pathway is particularly suitable for this task as myeloid or lymphoid cells express Fas only if activated, while FasL is preferentially expressed on activated CD4+ Th1 cells. Moreover, activated T cells eliminate themselves by the Fas-mediated pathway. Whether this happens by fratricide only, or also by suicide or both is open. Moreover, CD4+ CTL are particularly suitable for killing tumor cells as well, as they are efficient effectors in bystander lysis in contrast to CD8+ CTL. On the other hand, the non-specific killing via Fas-FasL interaction, which is an important reason for the bystander lysis, may have unwanted effects in that cells which should not be eliminated could be killed. Such reactions affecting various organs and cells, e.g. the liver, thyroid or islet cells of the pancreas could be an explanation for certain autoimmune diseases.
Animals↗
Analysis of the yeast transcription factor TFIIA: distinct functional regions and a polymerase II-specific role in basal and activated transcription.
To probe the structure and function of the Saccharomyces cerevisiae general transcription factor TFIIA, we have systematically mutagenized the genes encoding both subunits and analyzed the effects of the mutations both in vivo and in vitro. We found that the central nonconserved region of the large subunit is not essential for function and likely acts as a spacer between the conserved N- and C-terminal regions. Deletion mutagenesis of the large subunit defined a region which is required for TATA binding protein (TBP) interaction. Alanine scanning mutagenesis defined a cluster of four basic residues which are likely required for interaction with DNA in the TBP-DNA complex. Much of the conserved regions of both subunits is required for subunit association, suggesting that these conserved regions fold into compact domains which extensively interact. In vitro transcription performed with extracts from yeast strains with mutations in either the large or the small TFIIA subunit demonstrated that TFIIA stimulates both basal and activated polymerase II (Pol II) transcription. The TFIIA-depleted extracts have normal Pol I and Pol III transcription activity, showing that TFIIA is a Pol II-specific factor. In vivo depletion of TFIIA activity reduced transcription from four different Pol II promoters. Finally, alanine scanning mutagenesis of TFIIA's small subunit has identified at least one mutation which is defective in transcription but which is not defective in subunit association or binding to TBP or TBP-DNA complexes.
Alanine↗
Pathways of tumour cell killing by activated macrophages: both tumour cell membrane and nucleus can be the primary target.
Plasma membrane and nucleus can be primary targets of tumour cell killing by activated macrophages (AMø). Necrotic-type cytotoxicity with loss of membrane integrity and cytoplasmic swelling was expressed by AMø from normal and from perforin-deficient mice, indicating that perforin was not involved. Incubation with AMø consistently triggered the release of thymidine from prelabelled targets, whereas chromatin condensation and small DNA fragments were only occasionally detected. It is shown by means of Pulsed-Field Gel Electrophoresis that DNA degradation in target cells is a slowly progressing process that may stop at any time, indicating that nuclear-type killing doesnot necessarily lead to the formation of low molecular weight fragments. Neither Fas nor the p55 tumour necrosis factor receptor appear to be involved in signalling nuclear-type killing. Accordingly, AMø do mediate membrane- and nuclear-type killing but the mechanisms differ from those identified in T cell cytotoxicity.
Journal Article↗
Cytomegalovirus enteritis after treatment with 5-fluorouracil, leukovorin, cisplatin, and alpha-interferon.
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Antineoplastic Combined Chemotherapy Protocols↗
Functional hierarchy of AUUUA motifs in mediating rapid interleukin-3 mRNA decay.
In mast cells, expression of interleukin-3 (IL-3) is induced following IgE-receptor activation via calcium-dependent mRNA stabilization. We performed a mutational analysis of the 8 AUUUA motifs located in the 3'-untranslated region of IL-3 mRNA, and analyzed the effect on mRNA stability in PB-3c mast cells. Similar to endogenous IL-3 mRNA, exogenous IL-3 transcripts had a short half-life and were stabilized following stimulation of calcium influx by ionomycin. Specific mutation of 3 AUUUA motifs had almost the same stabilizing effect as deletion of the entire AU-rich region. Mutating even a single critical motif led to a weak, but significant mRNA stabilization. Ionomycin treatment did not further enhance the expression of mutationally stabilized transcripts. Our data fit a model, where within a cluster of 6 AUUUA motifs, 3 adjacent motifs are required for binding of a factor which mediates rapid IL-3 mRNA decay. Activity of such a factor might be under control of a calcium-dependent pathway.
Animals↗
Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism.
Basal transcription of many genes in yeast is repressed by Mot1, an essential protein which is a member of the Snf2/Swi2 family of conserved nuclear factors. ADI is an ATP-dependent inhibitor of TATA-binding protein (TBP) binding to DNA that inhibits transcription in vitro. Here we demonstrate that ADI is encoded by the MOT1 gene. Mutation of MOT1 abolishes ADI activity and derepresses basal transcription in vitro and in vivo. Recombinant Mot1 removes TBP from DNA and Mot1 contains an ATPase activity which is essential for its function. Genetic interactions between Mot1 and TBP indicate that their functions are interlinked in vivo. These results provide a general model for understanding the mechanism of action of a large family of nuclear factors involved in processes such as transcription and DNA repair.
Adenosine Triphosphatases↗
Cyclosporin A inhibits growth of autocrine tumour cell lines by destabilizing interleukin-3 mRNA.
In T cells, cyclosporin A (CsA) exerts its immunosuppressive effect by preventing transcriptional induction of the expression of interleukin(IL)-2. This is achieved by a mechanism that involves binding of a CsA-cyclophilin complex to calcineurin, which in turn inhibits the phosphatase-controlled translocation of transcription factor NFAT to the nucleus. We have previously identified IL-3 as an autocrine oncogenic regulator in tumour cell lines generated by introducing the v-H-ras oncogene into IL-3-dependent mast cells. Here we report that CsA specifically blocks autocrine tumour cell growth. The mechanism involves down-regulation of IL-3 expression by destabilization of the messenger RNA and requires ongoing transcription. Transcripts from exogenous IL-3 genes lacking the (A+U)-rich element (ARE) in the 3' untranslated terminal repeat could not be destabilized, suggesting that at least part of this sequence, which is known to mediate decay of short-lived mRNA, participates in a CsA-sensitive regulatory mechanism.
Animals↗
Fas antigen is the major target molecule for CD4+ T cell-mediated cytotoxicity.
Activation of the Fas cell surface molecule, either by specific antibody or by its as yet unidentified ligand, has been shown to induce apoptosis. Because apoptosis is also evoked in target cells by cytolytic T cells, we investigated whether the Fas pathway is involved in CD4+ T cell-mediated cytotoxicity. Analysis of Fas expression in APC, such as the B lymphoma A20.2J and MHC class II-transfected fibroblasts RT2.3, revealed a correlation between the degree of expression and sensitivity to cytotoxic attack, high level of Fas expression in A20.2J being associated with efficient lysis. To examine whether increased Fas expression in RT2.3 would render these cells more susceptible to CD4+ CTL lysis, they were transfected with a Fas gene expression vector. Indeed, Fas- but not mock-transfected RT2.3 proved to be more sensitive to lysis by either Ag specifically or nonspecifically activated CD4+ CTL. Similarly, MHC class II-negative, Fas-transfected L1210 leukemia cells were lysed with nonspecifically activated CD4+ CTL. The importance of the Fas engagement in CD4+ CTL-mediated cytotoxicity is further substantiated by the failure of both cloned and normal CD4+ CTL to lyse B cell blasts from Ipr mice. These mice are known to have a defect in functional Fas expression. Although the bulk of CD4+ T cell-mediated lysis appears to be Fas induced, the fact that the effector phase of A20.2J lysis is only partially Ca2+ independent indicates that other pathways also contribute to target cell death.
Animals↗