TATA Sequence Research Articles

Broad compatibility between yeast UAS elements and core promoters and identification of promoter elements that determine cofactor specificity

Three classes of yeast protein-coding genes are distinguished by their dependence on the transcription cofactors TFIID, SAGA, and Mediator (MED) Tail, but whether this dependence is determined by the core promoter, upstream activating sequences (UASs), or other gene features is unclear. Also unclear is whether UASs can broadly activate transcription from the different promoter classes. Here, we measure transcription and cofactor specificity for thousands of UAS-core promoter combinations and find that most UASs broadly activate promoters regardless of regulatory class, while few display strong promoter specificity. However, matching UASs and promoters from the same gene class is generally important for optimal expression. We find that sensitivity to rapid depletion of MED Tail or SAGA is dependent on the identity of both UAS and core promoter, while dependence on TFIID localizes to only the promoter. Finally, our results suggest the role of TATA and TATA-like promoter sequences in MED Tail function.

Phenotypic variability in expressivity and deleterious capability of the homozygous UGT1A1*28 genotype

The UGT1A1*28 polymorphism (rs3064744) is responsible for Gilbert's Syndrome (GS) and is characterized by allelic variability based on TA repeats in the TATA sequence of the UGT1A1 gene promoter, whose alleles 7TA and 8TA are associated with elevated bilirubin, as well as an increased risk of certain neoplasms and adverse reactions to the chemotherapy Irinotecan. In contrast, the existence of hyperbilirubinemia has been considered a preventive factor for certain cardiovascular and oncological pathologies. We analyzed the allelic distribution of the UGT1A1 allele in the Valencian population with hyperbilirubinemia and Valencian patients diagnosed with digestive neoplasms; we studied their biochemical profiles, specifically comparing individuals with the UGT1A1*28 genotype in homozygosis of both groups.

Molecular biology of the cell

Molecular biology of the cell

Unannotated single nucleotide polymorphisms in the TATA box of erythropoiesis genes show in vitro positive involvements in cognitive and mental disorders

BackgroundHemoglobin is a tetramer consisting of two α-chains and two β-chains of globin. Hereditary aberrations in the synthesis of one of the globin chains are at the root of thalassemia, one of the most prevalent monogenic diseases worldwide. In humans, in addition to α- and β-globins, embryonic zeta-globin and fetal γ-globin are expressed. Immediately after birth, the expression of fetal Aγ- and Gγ-globin ceases, and then adult β-globin is mostly expressed. It has been shown that in addition to erythroid cells, hemoglobin is widely expressed in nonerythroid cells including neurons of the cortex, hippocampus, and cerebellum in rodents; embryonic and adult brain neurons in mice; and mesencephalic dopaminergic brain cells in humans, mice, and rats. Lately, there is growing evidence that different forms of anemia (changes in the number and quality of blood cells) may be involved in (or may accompany) the pathogenesis of various cognitive and mental disorders, such as Alzheimer’s and Parkinson’s diseases, depression of various severity levels, bipolar disorders, and schizophrenia. Higher hemoglobin concentrations in the blood may lead to hyperviscosity, hypovolemia, and lung diseases, which may cause brain hypoxia and anomalies of brain function, which may also result in cognitive deficits.MethodsIn this study, a search for unannotated single-nucleotide polymorphisms (SNPs) of erythroid genes was initially performed using our previously created and published SNP-TATA_Z-tester, which is a Web service for computational analysis of a given SNP for in silico estimation of its influence on the affinity of TATA-binding protein (TBP) for TATA and TATA-like sequences. The obtained predictions were finally verified in vitro by an electrophoretic mobility shift assay (EMSA).ResultsOn the basis of these experimental in vitro results and literature data, we studied TATA box SNPs influencing both human erythropoiesis and cognitive abilities. For instance, TBP–TATA affinity in the HbZ promoter decreases 6.6-fold as a result of a substitution in the TATA box (rs113180943), thereby possibly disrupting stage-dependent events of “switching” of hemoglobin genes and thus causing erythroblastosis. Therefore, rs113180943 may be a candidate marker of severe hemoglobinopathies with comorbid cognitive and mental disorders associated with cerebral blood flow disturbances.ConclusionsThe literature data and experimental and computations results suggest that the uncovered candidate SNP markers of erythropoiesis anomalies may also be studied in cohorts of patients with cognitive and/or mental disorders with comorbid erythropoiesis diseases in comparison to conventionally healthy volunteers. Research into the regulatory mechanisms by which the identified SNP markers contribute to the development of hemoglobinopathies and of the associated cognitive deficits will allow physicians not only to take timely and adequate measures against hemoglobinopathies but also to implement strategies preventing cognitive and mental disorders.

Assembly of SNAPc, Bdp1, and TBP on the U6 snRNA Gene Promoter in Drosophila melanogaster.

U6 snRNA is transcribed by RNA polymerase III (Pol III) and has an external upstream promoter that consists of a TATA sequence recognized by the TBP subunit of the Pol III basal transcription factor IIIB and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). Previously, we found that Drosophila melanogaster SNAPc (DmSNAPc) bound to the U6 PSE can recruit the Pol III general transcription factor Bdp1 to form a stable complex with the DNA. Here, we show that DmSNAPc-Bdp1 can recruit TBP to the U6 promoter, and we identify a region of Bdp1 that is sufficient for TBP recruitment. Moreover, we find that this same region of Bdp1 cross-links to nucleotides within the U6 PSE at positions that also cross-link to DmSNAPc. Finally, cross-linking mass spectrometry reveals likely interactions of specific DmSNAPc subunits with Bdp1 and TBP. These data, together with previous findings, have allowed us to build a more comprehensive model of the DmSNAPc-Bdp1-TBP complex on the U6 promoter that includes nearly all of DmSNAPc, a portion of Bdp1, and the conserved region of TBP.

The DNA conformational energy landscape: sequence-dependent conformational equilibria of duplex DNA

The free energy surfaces of duplex dinucleotide steps were mapped in a principal conformational subspace derived from crystal structure data on DNA duplex oligomers. The three dimensional subspace, spanned by collective degrees of freedom representing linear combinations of the Cartesian coordinates of the backbone and sugar atoms of both strands accounted for 77% of the total variance of the observed structural distribution. The features of the subspace free energy surface correspond well to the distribution of observed structures exhibiting a clear separation of A- and B-family classes. The sequence dependence of the relative A / B-form conformational equilibria was derived from the corresponding subspace free energy surfaces at physiological conditions. A B-philicity scale representing the mole fraction of the BI-form vs the A-family for the 10 unique dinucleotide steps revealed three classes of sequences: highly B-philic (GC/GC & CG/CG), B-philic (AC/GT, AA/TT, AT/AT, CA/TG, AG/CT & GG/CC) and A-philic (GA/TC & TA/TA). The high propensity of the TA/TA step to adopt the A-form conformation is in accord with single crystal X-ray diffraction data and has biological significance in view of the frequent presence of the TATA sequence motif in transcriptional promoter regions.

PD-1 Imposes Qualitative Control of Cellular Transcriptomes in Response to T Cell Activation.

Targeted blockade of programmed cell death 1 (PD-1), an immune-checkpoint receptor that inhibits Tcell activation, provides clinical benefits in various cancers. However, how PD-1 modulates gene expression in Tcells remains enigmatic. Here weinvestigated how PD-1 affects transcriptome changes induced by Tcell receptor (TCR) activation. Intriguingly, we identified a huge variance in PD-1 sensitivity among TCR-inducible genes. When we quantified the half maximal effective concentration (EC50) as the relationship between change in gene expression and TCR signal strength, we found that genes associated with survival and proliferation were efficiently expressed upon TCR activation and resistant to PD-1-mediated inhibition. Conversely, genes encoding cytokines and effector molecules were expressed less efficiently and sensitive to PD-1-mediated inhibition. We further demonstrated that transcription factor binding motifs and CpG frequency in the promoter region affect EC50 and thus the PD-1 sensitivity of genes. Our findings explain how PD-1, dependent on the TCR signal strength, calibrates cellular transcriptomes to shape functional properties of Tcell populations.

Elucidating the Molecular Binding Mechanism of the Tata-Binding Protein using Pie-Pife

The TATA-box binding protein (TBP) plays an important regulatory role in regulating gene expression by mediating the formation of the pre-initiation complex on promotor DNA. When binding to DNA, TBP melts the double helix and forms a stable complex while bending the dsDNA by ∼90o along the TATA sequence. In vitro single molecule experiments based on single-pair Förster resonance energy transfer (FRET) have recently revealed a dynamic, step-wise binding process via different intermediate conformations of the dsDNA. In vivo, TBP exists in different forms, e.g. as dimerbound to the dsDNA, or as part of different co-activator/deactivator complexes in the subsequent steps of pre-initiation complex formation. These states however are only observed at cellular concentration of TBP in the micromolar range, which is too high for in vitro single molecule fluorescence imaging. In this contribution, we present a new strategy to visualizes unlabelled TBP binding on DNA with changes in DNA conformation at high concentrations: binding of TBP is monitored via protein-induced fluorescence enhancement (PIFE) while single-pair FRET reports on ongoing conformational changes in the DNA/TBP complex simultaneously. We combine this label-free PIFE-FRET approach with pulsed interleaved excitation (PIE) spectroscopy, to investigate conformational dynamics of freely diffusing protein-DNA complexes in solution at the focus of a confocal microscope. Without the need of surface immobilization it can overcome concentration limitations of multi-colour assays by monitoring two parameters, binding and conformation rearrangement at the same.

SAGA mediates transcription from the TATA-like element independently of Taf1p/TFIID but dependent on core promoter structures in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, core promoters of class II genes contain a TATA element, either a TATA box (TATA[A/T]A[A/T][A/G]) or TATA-like element (1 or 2 bp mismatched version of the TATA box). The TATA element directs the assembly of the preinitiation complex (PIC) to ensure accurate transcriptional initiation. It has been proposed the PIC is assembled by two distinct pathways in which TBP is delivered by TFIID or SAGA, leading to the widely accepted model that these complexes mediate transcription mainly from TATA-like element- or TATA box-containing promoters, respectively. Although both complexes are involved in transcription of nearly all class II genes, it remains unclear how efficiently SAGA mediates transcription from TATA-like element-containing promoters independently of TFIID. We found that transcription from the TATA box-containing AGP1 promoter was greatly stimulated in a Spt3p-dependent manner after inactivation of Taf1p/TFIID. Thus, this promoter provides a novel experimental system in which to evaluate SAGA-mediated transcription from TATA-like element(s). We quantitatively measured transcription from various TATA-like elements in the Taf1p-dependent CYC1 promoter and Taf1p-independent AGP1 promoter. The results revealed that SAGA could mediate transcription from at least some TATA-like elements independently of Taf1p/TFIID, and that Taf1p-dependence or -independence is highly robust with respect to variation of the TATA sequence. Furthermore, chimeric promoter mapping revealed that Taf1p-dependence or independence was conferred by the upstream activating sequence (UAS), whereas Spt3p-dependent transcriptional stimulation after inactivation of Taf1p/TFIID was specific to the AGP1 promoter and dependent on core promoter regions other than the TATA box. These results suggest that TFIID and/or SAGA are regulated in two steps: the UAS first specifies TFIID or SAGA as the predominant factor on a given promoter, and then the core promoter structure guides the pertinent factor to conduct transcription in an appropriate manner.

Molecular Mechanism of Mot1, a TATA-binding Protein (TBP)-DNA Dissociating Enzyme

The essential Saccharomyces cerevisiae ATPase Mot1 globally regulates transcription by impacting the genomic distribution and activity of the TATA-binding protein (TBP). In vitro, Mot1 forms a ternary complex with TBP and DNA and can use ATP hydrolysis to dissociate the TBP-DNA complex. Prior work suggested an interaction between the ATPase domain and a functionally important segment of DNA flanking the TATA sequence. However, how ATP hydrolysis facilitates removal of TBP from DNA is not well understood, and several models have been proposed. To gain insight into the Mot1 mechanism, we dissected the role of the flanking DNA segment by biochemical analysis of complexes formed using DNAs with short single-stranded gaps. In parallel, we used a DNA tethered cleavage approach to map regions of Mot1 in proximity to the DNA under different conditions. Our results define non-equivalent roles for bases within a broad segment of flanking DNA required for Mot1 action. Moreover, we present biochemical evidence for two distinct conformations of the Mot1 ATPase, the detection of which can be modulated by ATP analogs as well as DNA sequence flanking the TATA sequence. We also show using purified complexes that Mot1 dissociation of a stable, high affinity TBP-DNA interaction is surprisingly inefficient, suggesting how other transcription factors that bind to TBP may compete with Mot1. Taken together, these results suggest that TBP-DNA affinity as well as other aspects of promoter sequence influence Mot1 function in vivo.

Diorganotin(IV) complexes with 2-furancarboxylic acid hydrazone derivative of benzoylacetone: Synthesis, X-ray structure, antibacterial activity, DNA cleavage and molecular docking

Two new diorganotin(IV) complexes, Me2SnL and Ph2SnL, have been synthesized from the reaction of Me2SnCl2 and Ph2SnCl2 with the hydrazone H2L [H2L = (Furan-2-yl) (5-hydroxy-3-methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-methanone] derived from furan-2-carbohydrazide and benzoylacetone. The new compounds have been characterized by elemental and spectroscopic analyses. The crystal structures of the monohydrate form of the ligand and of the Me2SnL derivative have been also determined by X-ray crystallography. Experimental evidences confirm the existence of the hydrazone ligand exclusively in cyclic form in both solution and solid state. On coordination to tin the hydrazone undergoes a ring opening reaction and a doubly deprotonation to act as a tridentate ligand via imine nitrogen and enolic oxygens. The tin atom in the complexes is five coordinate with geometry between square-pyramidal and trigonal-bipyramidal. The in vitro antibacterial activity of ligand and its complexes has been evaluated against Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. The interaction between compounds with bacterial DNA was also studied by molecular docking. Our findings indicate that diphenyltin(IV) complex, by binding to DNA via minor groove to TATA sequence in genes upstream, has good activities along with the standard antibacterial drugs. Our agarose-gel electrophoresis experiments show that the ligand exert DNA cleavage, while Me2SnL and Ph2SnL did not.

A Random Screen Using a Novel Reporter Assay System Reveals a Set of Sequences That Are Preferred as the TATA or TATA-Like Elements in the CYC1 Promoter of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the core promoters of class II genes contain either TATA or TATA-like elements to direct accurate transcriptional initiation. Genome-wide analyses show that the consensus sequence of the TATA element is TATAWAWR (8 bp), whereas TATA-like elements carry one or two mismatches to this consensus. The fact that several functionally distinct TATA sequences have been identified indicates that these elements may function, at least to some extent, in a gene-specific manner. The purpose of the present study was to identify functional TATA sequences enriched in one particular core promoter and compare them with the TATA or TATA-like elements that serve as the pre-initiation complex (PIC) assembly sites on the yeast genome. For this purpose, we conducted a randomized screen of the TATA element in the CYC1 promoter by using a novel reporter assay system and identified several hundreds of unique sequences that were tentatively classified into nine groups. The results indicated that the 7 bp TATA element (i.e., TATAWAD) and several sets of TATA-like sequences are preferred specifically by this promoter. Furthermore, we find that the most frequently isolated TATA-like sequence, i.e., TATTTAAA, is actually utilized as a functional core promoter element for the endogenous genes, e.g., ADE5,7 and ADE6. Collectively, these results indicate that the sequence requirements for the functional TATA or TATA-like elements in one particular core promoter are not as stringent. However, the variation of these sequences differs significantly from that of the PIC assembly sites on the genome, presumably depending on promoter structures and reflecting the gene-specific function of these sequences.

Association of polymorphism within LTF gene promoter with lactoferrin concentration in milk of Holstein cows.

This study analyzed the association between single nucleotide polymorphism (A/C) in position -28 located in the TATA box of LTF gene and the lactoferrin concentration in bovine milk secreted by healthy and infected udders. Out of 241, 69 cows were selected into the experimental group and were divided into 3 groups according to mean value of somatic cell count (SCC): I < 180,000 cells/mL, II: 180,000-350,000 cells/mL and III > 350,000 cells/mL. In each SCC group, three LTF genotypes: AA, AC and CC were identified by PCR-SSCP method. A total of 604 milk samples were collected monthly and lactoferrin concentration was measured by ELISA. The 1-way ANOVA within SCC groups was performed to estimate association of -28 A/C genotypes with mean lactoferrin concentration per lactation. In the group of healthy cows (< 180,000 cells/mL) LTF concentration in milk cows with the AA genotype (107.58 ± 17.92 μg/mL) was significantly higher than in homozygotes CC (52.09 ± 19.01 μg/mL). Unexpectedly, in cows with elevated SCC (> 350,000 cells/mL) we observed a significant opposite relationship (207.21 ± 28.50 in CC vs 115.0 ± 28.6 μg/mL in AA). We hypothesized that a promoter with allele C, which cannot be recognized as a TATA sequence is becoming more accessible for other transcription factors, which may induce alternative LTF gene expression. We assume that our results demonstrate a very interesting effect of differential gene expression depending on polymorphism in a key regulatory motif (TATA box) and also on the health status of mammary tissues.

Heart-specific expression of FGF-16 and a potential role in postnatal cardioprotection

Fibroblast growth factor 16 (FGF-16) was originally cloned from rat heart. Subsequent investigation of mouse FGF-16, including generation of null mice, revealed a specific pattern of expression in the endocardium and epicardium, and role for FGF-16 during embryonic heart development. FGF-16 is expressed mainly in brown adipose tissue during rat embryonic development, but is expressed mainly in the murine heart after birth. There is also an apparent switch from limited endocardial and epicardial expression in the embryo to the myocardium in the perinatal period. The FGF-16 gene and its location on the X chromosome are conserved between human and murine species, and no other member of the FGF family shows this pattern of spatial and temporal expression. The human and murine FGF-16 gene promoter regions also share an equivalent location for TATA sequences, as well as adjacent putative binding sites for transcription factors linked to cardiac expression and response to stress. Recent evidence has implicated nonsense mutation of FGF-16 with increased cardiovascular risk, and FGF-16 supplementation with cardioprotection. Here we review the important role of FGF-16 in embryonic heart development, its gene regulation, and evidence for FGF-16 as an endogenous and exogenous cardiac-specific and protective factor in the postnatal heart. Moreover, given the conservation of the FGF-16 gene and its chromosomal location between species, the question of support for a cardiac role in the human population is also considered.

Direct observation of preferential processing of clustered abasic DNA damages with APE1 in TATA box and CpG island by reaction kinetics and fluorescence dynamics

Sequences like the core element of TATA box and CpG island are frequently encountered in the genome and related to transcription. The fate of repair of clustered abasic sites in such sequences of genomic importance is largely unknown. This prompted us to investigate the sequence dependence of cleavage efficiency of APE1 enzyme at abasic sites within the core sequences of TATA box and CpG island using fluorescence dynamics and reaction kinetics. Simultaneous molecular dynamics study through steady state and time resolved fluorescence spectroscopy using unique ethidium bromide dye release assay confirmed an elevated amount of abasic site cleavage of the TATA box sequence as compared to the core CpG island. Reaction kinetics showed that catalytic efficiency of APE1 for abasic site cleavage of core CpG island sequence was ∼4 times lower as compared to that of the TATA box. Higher value of Km was obtained from the core CpG island sequence than the TATA box sequence. This suggests a greater binding effect of APE1 enzyme on TATA sequence that signifies a prominent role of the sequence context of the DNA substrate. Evidently, a faster response from APE1 was obtained for clustered abasic damage repair of TATA box core sequences than CpG island consensus sequences. The neighboring bases of the abasic sites in the complementary DNA strand were found to have significant contribution in addition to the flanking bases in modulating APE1 activity. The repair refractivity of the bistranded clustered abasic sites arise from the slow processing of the second abasic site, consequently resulting in decreased overall production of potentially lethal double strand breaks.

Cloning and Analysis of the RNA Polymerase i-Promoter of Nicotiana benthaminana

Promoter is an important transcriptional regulatory element,which controls the levels and patterns of gene expression. RNA Pol I is a cellular enzyme that is abundantly expressed in cells and transcribes rRNA precursor lacking a 5' cap,a 3' poly( A) tail and introns. Thus,viral RNA synthesized in cells transfected with Pol I-driven plasmids containing viral genomic cDNA has precise sequences. This may increase the expression level and biological safety of the plant RNA-based expression system. In addition,Nicotiana benthaminana is a kind of model organism which widely used in plant bioreactor and plant pathology. However, there is no nucleic acid sequence data about its RNA Pol I promoter for now. Therefore,Nicotiana benthaminana Pol I promoter sequence we cloned and analysized its transcription initiation site( TIS). The 514 bp RNA pol I promoter sequence( KC352713) was amplified by semi-net PCR form Nicotiana benthaminana and the third A residue in the core sequence TATA( G) TA( N) GGGGG was predicted to be the transcription initiation site( TIS) by comparison of sequences flanking RNA pol I promoter TIS in silicon. The predicted result was conformed by the 5' RACE and sequence analysis. This result will be beneficial for the further research of both Pol I promoter and Pol I-based plant virus expression vectors.

Characterization of sea bass FSHβ 5′ flanking region: transcriptional control by 17β-estradiol

The sea bass follicle-stimulating hormone 5' flanking region (sbFSHβ 5' FR) was cloned and characterized in order to study the molecular mechanisms underlying transcriptional regulation of the sbFSHβ gene. Analysis of the ~3.5kb of this region revealed the presence of several putative cis-acting elements, including steroid hormone response elements, cAMP response elements, pituitary-specific transcription factor response elements, activator protein-1 response elements and TATA sequence. Deleted constructs containing ~3.5kb of the sbFSHβ 5' FR fused to a luciferase reporter gene were transiently transfected into human embryonic kidney (HEK 293) and mouse mature gonadotrope (LβT2) cell lines. The sbFSHβ 5' FR was efficiently expressed under basal conditions in LβT2 but not in HEK 293, pointing to both positive and negative regulatory elements. In order to elucidate the estrogen-mediated sbFSHβ transcriptional activity, in vitro treatments with 17β-estradiol were carried out on primary cultures of pituitary cells and LβT2 cells transiently expressing luciferase under the control of sbFSHβ 5' FR. Overall, these results demonstrate that 17β-estradiol inhibits sbFSHβ gene expression directly at the level of the pituitary. However, it was also shown that estrogen did not induce changes of the sbFSH promoter-directed luciferase activity, suggesting that sbFSHβ 5'FR (~3.5kb) activity is cell type dependent and its estrogen regulation could require cis-acting elements located upstream of the promoter region, which is characterized in this article.

Nuclear factor Y (NF-Y) regulates the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes

Type XI collagen, a heterotrimer composed of α1(XI), α2(XI), and α3(XI), plays a critical role in cartilage formation and in skeletal morphogenesis. However, the transcriptional regulation of α1(XI) collagen gene (Col11a1) in chondrocyte is poorly characterized. In this study, we investigated the proximal promoter of mouse Col11a1 gene in chondrocytes. Major transcription start site was located at -299bp upstream of the translation start site, and the proximal promoter lacks a TATA sequence but has a high guanine-cytosine (GC) content. Cell transfection experiments demonstrated that the segment from -116 to -256 is necessary for activation of the proximal Col11a1 promoter, and an electrophoretic mobility shift assay showed that a nuclear protein is bound to the segment from -116 to -176 in this promoter. Additional comparative and in silico analyses demonstrated that an ATTGG sequence, which is critical for binding to nuclear factor Y (NF-Y), is within the highly conserved region from -135 to -145. Interference assays using wild-type and mutant oligonucleotide or with specific antibody revealed that NF-Y protein is bound to this region. Cell transfection experiments with reporter constructs in the absence of NF-Y binding sequence exhibited the suppression of the promoter activity. Furthermore, chromatin immunoprecipitation assay demonstrated that NF-Y protein is directly bound to this region in vivo, and overexpression of dominant-negative NF-Y A mutant also inhibited the proximal promoter activity. Taken together, these results indicate that the transcription factor NF-Y regulates the proximal promoter activity of mouse Col11a1 gene in chondrocytes.

Genomic organization of the cytosolic manganese superoxide dismutase gene from the Pacific white shrimp, Litopenaeus vannamei, and its response to thermal stress

Cytosolic manganese superoxide dismutase (cMnSOD) is an important antioxidant enzyme which catalyzes the conversion of superoxides to oxygen and hydrogen peroxide in several organisms. In the Pacific white shrimp, Litopenaeus vannamei, three cMnSOD genes (LvcMnSOD1-3) have previously been characterized. Here, the genomic structure of LvcMnSOD2 and its mRNA expression in response to thermal stress was examined. Analysis of the nucleotide sequence demonstrated that LvcMnSOD2 is comprised of 2392 bp spanning from the ATG translation start site to the stop codon and contains six exons interrupted by five introns. The 5′ region upstream of the LvcMnSOD2 gene contains several putative regulatory elements but lacks the accepted TATA sequence. The putative transcription factor binding elements that may be involved in LvcMnSOD2 mRNA expression level include activator protein-1 (AP-1), cAMP response element binding protein (CREB), upstream stimulatory factor (USF), CAAT-enhancer binding protein (C/EBP), nuclear factor-κB (NF-κB) and heat shock regulatory element (HSE). In addition, we compared the 5′ upstream sequences of the LvcMnSOD2 gene between two shrimp strains that are resistant or susceptible to Taura syndrome virus (TSV), respectively, which revealed the absence of the USF and C/EBP elements at positions −2125 and −1986, respectively, in the TSV-susceptible shrimp line. Moreover, genomic variations between the two shrimp strains were detected in some of the putative C/EBP, USF, HSE and NF-κB transcription factor binding elements. That these genomic variations might be involved in the TSV resistance as well as in stress responses remains to be evaluated. The presence of 15 putative HSEs suggests that the expression of LvcMnSOD2 is regulated under thermal stress. Here, we found that in response to a 1 or 3 h thermal stress (35 °C), the mRNA expression levels of LvcMnSOD2 were significantly increased and then gradually decreased in the recovering phase at room temperature (25 °C) to control levels by 3 h after the heat shock. Thus, the antioxidant system may be induced to protect cells from the oxidative damage caused by thermal stress. The genomic organization of LvcMnSOD2 likely provides a clue to the mechanisms that might regulate the antioxidant defense pathway in shrimps and so potentially in marine invertebrates.

Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

Epstein-Barr virus (EBV) expresses an immediate-early protein, Rta, to activate the transcription of EBV lytic genes. This protein usually binds to Rta-response elements or interacts with Sp1 or Zta via a mediator protein, MCAF1, to activate transcription. Rta is also known to interact with TBP and TFIIB to activate transcription. This study finds that Rta interacts with TAF4, a component of TFIID complex, in vitro and in vivo, and on the TATA sequence in the BcLF1 promoter. Rta also interacts with TAF4 and Sp1 on Sp1-binding sequences on TATA-less promoters, including those of BNLF1, BALF5, and the human androgen receptor. These interactions are important to the transcriptional activation of these genes by Rta since introducing TAF4 shRNA substantially reduces the ability of Rta to activate these promoters. This investigation reveals how Rta interacts with TFIID to stimulate transcription.