Showing papers on "Transcription (biology) published in 1998"
TL;DR: This approach extends the power of FISH to yield quantitative molecular information on a single cell by positioning probes along the transcription unit to determine the rates of transcription initiation and termination and messenger RNA processing.
Abstract: Fluorescence in situ hybridization (FISH) and digital imaging microscopy were modified to allow detection of single RNA molecules. Oligodeoxynucleotide probes were synthesized with five fluorochromes per molecule, and the light emitted by a single probe was calibrated. Points of light in exhaustively deconvolved images of hybridized cells gave fluorescent intensities and distances between probes consistent with single messenger RNA molecules. Analysis of beta-actin transcription sites after serum induction revealed synchronous and cyclical transcription from single genes. The rates of transcription initiation and termination and messenger RNA processing could be determined by positioning probes along the transcription unit. This approach extends the power of FISH to yield quantitative molecular information on a single cell.
1,299 citations
TL;DR: Injection of viruses into the rat brain showed that a SIN vector containing the green fluorescent protein gene under the control of the internal CMV promoter transduced neurons as efficiently as a wild-type vector, indicating that the HIV-1 LTR promoter is transcriptionally active in neurons even in the absence of Tat.
Abstract: We have constructed a new series of lentivirus vectors based on human immunodeficiency virus type 1 (HIV-1) that can transduce nondividing cells. The U3 region of the 5′ long terminal repeat (LTR) in vector constructs was replaced with the cytomegalovirus (CMV) promoter, resulting in Tat-independent transcription but still maintaining high levels of expression. A self-inactivating (SIN) vector was constructed by deleting 133 bp in the U3 region of the 3′ LTR, including the TATA box and binding sites for transcription factors Sp1 and NF-κB. The deletion is transferred to the 5′ LTR after reverse transcription and integration in infected cells, resulting in the transcriptional inactivation of the LTR in the proviruses. SIN viruses can be generated with no significant decreases in titer. Injection of viruses into the rat brain showed that a SIN vector containing the green fluorescent protein gene under the control of the internal CMV promoter transduced neurons as efficiently as a wild-type vector. Interestingly, a wild-type vector without an internal promoter also successfully transduced neurons in the brain, indicating that the HIV-1 LTR promoter is transcriptionally active in neurons even in the absence of Tat. Furthermore, injection of viruses into the subretinal space of the rat eye showed that wild-type vector transduced predominantly retinal pigment epithelium and photoreceptor cells, while SIN vector was able to transduce other types of retinal cells, including bipolar, Muller, horizontal, and amacrine cells. This finding suggests that the HIV-1 LTR can negatively influence the internal CMV promoter in some cell types. SIN HIV vectors should be safer for gene therapy, and they also have broader applicability as a means of high-level gene transfer and expression in nondividing cells.
1,254 citations
TL;DR: It is suggested that DNA damage enhances p53 activity as a transcription factor in part through carboxy-terminal acetylation that, in turn, is directed by amino- terminal phosphorylation.
Abstract: Activation of p53-mediated transcription is a critical cellular response to DNA damage. p53 stability and site-specific DNA-binding activity and, therefore, transcriptional activity, are modulated by post-translational modifications including phosphorylation and acetylation. Here we show that p53 is acetylated in vitro at separate sites by two different histone acetyltransferases (HATs), the coactivators p300 and PCAF. p300 acetylates Lys-382 in the carboxy-terminal region of p53, whereas PCAF acetylates Lys-320 in the nuclear localization signal. Acetylations at either site enhance sequence-specific DNA binding. Using a polyclonal antisera specific for p53 that is phosphorylated or acetylated at specific residues, we show that Lys-382 of human p53 becomes acetylated and Ser-33 and Ser-37 become phosphorylated in vivo after exposing cells to UV light or ionizing radiation. In vitro, amino-terminal p53 peptides phosphorylated at Ser-33 and/or at Ser-37 differentially inhibited p53 acetylation by each HAT. These results suggest that DNA damage enhances p53 activity as a transcription factor in part through carboxy-terminal acetylation that, in turn, is directed by amino-terminal phosphorylation.
1,214 citations
TL;DR: A point mutation A458T is introduced into the GR by gene targeting using the Cre/loxP system, impairs dimerization and therefore GRE-dependent transactivation while functions that require cross-talk with other transcription factors, such as transrepression of AP-1-driven genes, remain intact.
1,095 citations
TL;DR: Based on the observations summarized above, the mechanism of transcriptional activation by Smads is most likely defined by the combined requirements for interactions with other transcription factors and with promoter DNA sequences, which has a double DNA sequence requirement.
993 citations
TL;DR: Modeling the data suggests that high loads may halt RNAP by promoting a structural change which moves all or part of the enzyme backwards through a comparatively large distance, corresponding to 5 to 10 base pairs, contrasts with previous models that assumed force acts directly upon a single-base translocation step.
Abstract: RNA polymerase (RNAP) moves along DNA while carrying out transcription, acting as a molecular motor. Transcriptional velocities for single molecules of Escherichia coli RNAP were measured as progressively larger forces were applied by a feedback-controlled optical trap. The shapes of RNAP force-velocity curves are distinct from those of the motor enzymes myosin or kinesin, and indicate that biochemical steps limiting transcription rates at low loads do not generate movement. Modeling the data suggests that high loads may halt RNAP by promoting a structural change which moves all or part of the enzyme backwards through a comparatively large distance, corresponding to 5 to 10 base pairs. This contrasts with previous models that assumed force acts directly upon a single-base translocation step.
874 citations
TL;DR: Current models regarding the role of enhancers in the regulation of transcription by RNA polymerase II are presented.
Abstract: In eukaryotes, transcription of genes by RNA polymerase II yields messenger RNA intermediates from which protein products are synthesized. Transcriptional enhancers are discrete DNA elements that contain specific sequence motifs with which DNA-binding proteins interact and transmit molecular signals to genes. Here, current models regarding the role of enhancers in the regulation of transcription by RNA polymerase II are presented.
833 citations
TL;DR: MOP3 mRNA expression overlaps in a number of tissues with each of its four potential partner molecules in vivo and is demonstrated that MOP3 interacts with MOP4, CLOCK, hypoxia-inducible factor 1alpha (HIF1alpha), and HIF2alpha.
Abstract: We report that MOP3 is a general dimerization partner for a subset of the basic-helix–loop–helix (bHLH)-PER–ARNT–SIM (PAS) superfamily of transcriptional regulators. We demonstrated that MOP3 interacts with MOP4, CLOCK, hypoxia-inducible factor 1α (HIF1α), and HIF2α. A DNA selection protocol revealed that the MOP3-MOP4 heterodimer bound a CACGTGA-containing DNA element. Transient transfection experiments demonstrated that the MOP3-MOP4 and MOP3-CLOCK complexes bound this element in COS-1 cells and drove transcription from a linked luciferase reporter gene. We also deduced the high-affinity DNA binding sites for MOP3-HIF1α complex (TACGTGA) and used transient transfection experiments to demonstrate that the MOP3-HIF1α and MOP3-HIF2α heterodimers bound this element, drove transcription, and responded to cellular hypoxia. Finally, we found that MOP3 mRNA expression overlaps in a number of tissues with each of its four potential partner molecules in vivo.
760 citations
TL;DR: It is observed by in situ hybridization that dsRNA-mediated interference produced a substantial, although not complete, reduction in accumulation of nascent transcripts in the nucleus, while cytoplasmic accumulation of transcripts was virtually eliminated, indicating that the endogenous mRNA is the target for interference and suggesting a mechanism that degrades the targeted RNA before translation can occur.
Abstract: Introduction of exogenous double-stranded RNA (dsRNA) into Caenorhabditis elegans has been shown to specifically and potently disrupt the activity of genes containing homologous sequences. In this study we present evidence that the primary interference effects of dsRNA are post-transcriptional. First, we examined the primary DNA sequence after dsRNA-mediated interference and found no evidence for alterations. Second, we found that dsRNA-mediated interference with the upstream gene in a polar operon had no effect on the activity of the downstream gene; this finding argues against an effect on initiation or elongation of transcription. Third, we observed by in situ hybridization that dsRNA-mediated interference produced a substantial, although not complete, reduction in accumulation of nascent transcripts in the nucleus, while cytoplasmic accumulation of transcripts was virtually eliminated. These results indicate that the endogenous mRNA is the target for interference and suggest a mechanism that degrades the targeted RNA before translation can occur. This mechanism is not dependent on the SMG system, an mRNA surveillance system in C. elegans responsible for targeting and destroying aberrant messages. We suggest a model of how dsRNA might function in a catalytic mechanism to target homologous mRNAs for degradation.
730 citations
TL;DR: The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo.
Abstract: We report the identification of a transcription elongation factor from HeLa cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in conjunction with the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB). This factor, termed DRB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8. DSIF has been purified and is composed of 160-kD (p160) and 14-kD (p14) subunits. Isolation of a cDNA encoding DSIF p160 shows it to be a homolog of the Saccharomyces cerevisiae transcription factor Spt5. Recombinant Supt4h protein, the human homolog of yeast Spt4, is functionally equivalent to DSIF p14, indicating that DSIF is composed of the human homologs of Spt4 and Spt5. In addition to its negative role in elongation, DSIF is able to stimulate the rate of elongation by RNA Pol II in a reaction containing limiting concentrations of ribonucleoside triphosphates. A role for DSIF in transcription elongation is further supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG. The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast, also in this issue, indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo.
728 citations
TL;DR: The identification of these limiting co-repressor and co-activator complexes and their specific interaction motifs, in concert with solution of the structures of the receptor ligand-binding domain in apo (empty) and ligand bound forms, indicates a common molecular mechanism by which these factors activate and repress gene transcription.
TL;DR: The molecular mechanisms underlying the acetylation effects on chromatin condensation were investigated by analyzing the ability of differentially acetylated nucleosomal arrays to fold and oligomerize and yielded new insight into the molecular basis of acetylations effects on both transcription and higher-order compaction of nucleosomes.
Abstract: We have examined the effects of core histone acetylation on the transcriptional activity and higher-order folding of defined 12-mer nucleosomal arrays. Purified HeLa core histone octamers containing an average of 2, 6, or 12 acetates per octamer (8, 23, or 46% maximal site occupancy, respectively) were assembled onto a DNA template consisting of 12 tandem repeats of a 208-bp Lytechinus 5S rRNA gene fragment. Reconstituted nucleosomal arrays were transcribed in a Xenopus oocyte nuclear extract and analyzed by analytical hydrodynamic and electrophoretic approaches to determine the extent of array compaction. Results indicated that in buffer containing 5 mM free Mg2+ and 50 mM KCl, high levels of acetylation (12 acetates/octamer) completely inhibited higher-order folding and concurrently led to a 15-fold enhancement of transcription by RNA polymerase III. The molecular mechanisms underlying the acetylation effects on chromatin condensation were investigated by analyzing the ability of differentially acetylated nucleosomal arrays to fold and oligomerize. In MgCl2-containing buffer the folding of 12-mer nucleosomal arrays containing an average of two or six acetates per histone octamer was indistinguishable, while a level of 12 acetates per octamer completely disrupted the ability of nucleosomal arrays to form higher-order folded structures at all ionic conditions tested. In contrast, there was a linear relationship between the extent of histone octamer acetylation and the extent of disruption of Mg2+-dependent oligomerization. These results have yielded new insight into the molecular basis of acetylation effects on both transcription and higher-order compaction of nucleosomal arrays.
TL;DR: An immediate-early gene encoded by Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesv virus eight (HHV8) that activates lytic cycle gene expression from the latent viral genome is identified.
Abstract: Herpesviruses exist in two states, latency and a lytic productive cycle. Here we identify an immediate-early gene encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus eight (HHV8) that activates lytic cycle gene expression from the latent viral genome. The gene is a homologue of Rta, a transcriptional activator encoded by Epstein–Barr virus (EBV). KSHV/Rta activated KSHV early lytic genes, including virus-encoded interleukin 6 and polyadenylated nuclear RNA, and a late gene, small viral capsid antigen. In cells dually infected with Epstein–Barr virus and KSHV, each Rta activated only autologous lytic cycle genes. Expression of viral cytokines under control of the KSHV/Rta gene is likely to contribute to the pathogenesis of KSHV-associated diseases.
TL;DR: It is reported here that insulin shares with hypoxia the ability to induce the HIF‐1α/ARNT transcription complex in various cell types, and a novel signal transduction pathway of insulin and IGF‐I is established.
Abstract: Hypoxic stress induces the expression of genes associated with increased energy flux, including the glucose transporters Glut1 and Glut3, several glycolytic enzymes, nitric oxide synthase, tyrosine hydroxylase, erythropoietin and vascular endothelial growth factor (VEGF). Induction of these genes is mediated by a common basic helix-loop-helix-PAS transcription complex, the hypoxia-inducible factor-1alpha (HIF-1alpha)/aryl hydrocarbon nuclear translocator (ARNT). Insulin also induces some of these genes; however, the underlying mechanism is unestablished. We report here that insulin shares with hypoxia the ability to induce the HIF-1alpha/ARNT transcription complex in various cell types. This induction was demonstrated by electrophoretic mobility shift of the hypoxia response element (HRE), and abolished by specific antisera to HIF-1alpha and ARNT, and by transcription activation of HRE reporter vectors. Furthermore, basal and insulin-induced expression of Glut1, Glut3, aldolase A, phosphoglycerate kinase and VEGF was reduced in cells having a defective ARNT. Similarly, the insulin-induced activation of HRE reporter vectors and VEGF was impaired in these cells and was rescued by re-introduction of ARNT. Finally, insulin-like growth factor-I (IGF-I) also induced the HIF-1alpha/ARNT transcription complex. These observations establish a novel signal transduction pathway of insulin and IGF-I and broaden considerably the scope of activity of HIF-1alpha/ARNT.
TL;DR: The present review describes the topoisomerase I catalytic mechanisms with particular emphasis on the cleavage complex that represents the enzyme's catalytic intermediate and the site of action for camptothecins.
Journal Article•
TL;DR: This MMP-1 polymorphism contributes to increased transcription, and cells expressing the 2 G polymorphism may provide a mechanism for more aggressive matrix degradation, thereby facilitating cancer progression.
Abstract: Matrix metalloproteinases (MMPs) facilitate cellular invasion by degrading the extracellular matrix, and their regulation is partially dependent on transcription. Binding sites for members of the Ets family of transcription factors are present within MMP promoters and are potent positive regulators. We report a single nucleotide polymorphism at -1607 bp in the MMP-1 promoter, where an additional guanine (G) creates an Ets binding site, 5′-GGA-3′. This polymorphism displays significantly higher transcription in normal fibroblasts and in melanoma cells than the 1 G polymorphism, and it binds substantially more nuclear extract and recombinant ETS-1. Analysis of control DNAs from the Center d'Etude du Polymorphisme Humain pedigrees reveals that this polymorphism is not a mutation, with a frequency of the 2 G polymorphism at 30%. In contrast, in eight tumor cell lines, this frequency increased to 62.5% ( P < 0.0001). Thus, this MMP-1 polymorphism contributes to increased transcription, and cells expressing the 2 G polymorphism may provide a mechanism for more aggressive matrix degradation, thereby facilitating cancer progression.
TL;DR: The coactivator functions and physical interactions of Smad4 and CBP/p300 with Smad3 allow a model for the induction of gene expression in response to TGF-beta.
Abstract: Smads regulate transcription of defined genes in response to TGF-β receptor activation, although the mechanisms of Smad-mediated transcription are not well understood. We demonstrate that the TGF-β-inducible Smad3 uses the tumor suppressor Smad4/DPC4 and CBP/p300 as transcriptional coactivators, which associate with Smad3 in response to TGF-β. The association of CBP with Smad3 was localized to the carboxyl terminus of Smad3, which is required for transcriptional activation, and a defined segment in CBP. Furthermore, CBP/p300 stimulated both TGF-β- and Smad-induced transcription in a Smad4/DPC4-dependent fashion. Smad3 transactivation and TGF-β-induced transcription were inhibited by expressing E1A, which interferes with CBP functions. The coactivator functions and physical interactions of Smad4 and CBP/p300 with Smad3 allow a model for the induction of gene expression in response to TGF-β.
TL;DR: A mechanism whereby Smad2 and Smad3 positively and negatively regulate a TGF beta/activin target gene is described, which may prevent transcription by altering the conformation of the DNA-binding complex.
TL;DR: It is proposed that DsrA pairing stimulates RpoS translation by acting as an anti-antisense RNA, freeing the translation initiation region from the cis-acting antisense RNA and allowing increased translation.
Abstract: DsrA RNA regulates both transcription, by overcoming transcriptional silencing by the nucleoid-associated H-NS protein, and translation, by promoting efficient translation of the stress σ factor, RpoS. These two activities of DsrA can be separated by mutation: the first of three stem-loops of the 85 nucleotide RNA is necessary for RpoS translation but not for anti-H-NS action, while the second stem-loop is essential for antisilencing and less critical for RpoS translation. The third stem-loop, which behaves as a transcription terminator, can be substituted by the trp transcription terminator without loss of either DsrA function. The sequence of the first stem-loop of DsrA is complementary with the upstream leader portion of rpoS messenger RNA, suggesting that pairing of DsrA with the rpoS message might be important for translational regulation. Mutations in the Rpos leader and compensating mutations in DsrA confirm that this predicted pairing is necessary for DsrA stimulation of RpoS translation. We propose that DsrA pairing stimulates RpoS translation by acting as an anti-antisense RNA, freeing the translation initiation region from the cis-acting antisense RNA and allowing increased translation.
Journal Article•
TL;DR: The tissue-specific and gestational-age dependent expression of hTERT mRNA seen in human development suggests the presence of at least two regulatory mechanisms controlling the activity of telomerase: transcriptional control of the h TERT gene and alternate splicing of hterT transcripts.
Abstract: The correlation between telomerase activity, telomere lengths, and cellular replicative capacity has led to the theory that maintenance of telomere lengths by telomerase acts as a molecular clock to control replicative capacity and senescence. Regulation of this molecular clock may have applications in the treatment of cell aging and tumorigenesis, although little is presently known about the regulation of telomerase activity. To investigate possible mechanisms of regulation, we examined telomerase activity and the expression of the human telomerase RNA subunit and the human telomerase reverse transcriptase protein (hTERT) during human fetal heart, liver, and kidney development. The human telomerase RNA subunit is expressed in all three tissues at all gestational ages examined. hTERT expression correlates with telomerase activity in the liver, where both are expressed at all ages surveyed, and in the heart, where both are present until the 11th gestational week but not thereafter. However, although telomerase activity in the kidney is suppressed after the 15th gestational week, the hTERT transcript can be detected until at least the 21st week. Reverse transcription-PCR using primers within the reverse transcriptase domain of hTERT show the presence of multiple alternately spliced transcripts in these tissues, corresponding to full-length message as well as spliced messages with critical reverse transcriptase motifs deleted. Of note, telomerase activity in the kidney is only present at those gestational ages when full-length hTERT message is expressed (until approximately week 15), with spliced transcripts continuing to be expressed at later stages of development. The tissue-specific and gestational-age dependent expression of hTERT mRNA seen in human development suggests the presence of at least two regulatory mechanisms controlling the activity of telomerase: transcriptional control of the hTERT gene and alternate splicing of hTERT transcripts.
TL;DR: In this paper, a 2.7-A-resolution crystal structure of the DNA-binding domains of NFAT, Fos and Jun, in a quaternary complex with a DNA fragment containing the distal antigen-receptor response element from the interleukin-2 gene promoter, shows an extended interface between NFAT and AP-1.
Abstract: The nuclear factor of activated T cells (NFAT) and the AP-1 heterodimer, Fos-Jun, cooperatively bind a composite DNA site and synergistically activate the expression of many immune-response genes. A 2.7-A-resolution crystal structure of the DNA-binding domains of NFAT, Fos and Jun, in a quaternary complex with a DNA fragment containing the distal antigen-receptor response element from the interleukin-2 gene promoter, shows an extended interface between NFAT and AP-1, facilitated by the bending of Fos and DNA. The tight association of the three proteins on DNA creates a continuous groove for the recognition of 15 base pairs.
TL;DR: This work has shown that transcriptional regulators may be modified in an allosteric manner by response elements themselves to generate the pattern of regulation that is appropriate to an individual gene.
Abstract: Selective gene transcription is mediated in part by regulatory proteins that bind to DNA response elements These regulatory proteins receive global information from signal-transduction events But transcriptional regulators may also be modified in an allosteric manner by response elements themselves to generate the pattern of regulation that is appropriate to an individual gene
TL;DR: BRCA1 increases p53-dependent transcription from the p21WAF1/CIP1 and bax promoters and interacts with p53 proteins both in vitro and in vivo, indicating that BRCA 1 and p53 may coordinately regulate gene expression in their role as tumor suppressors.
Abstract: Mutations of the BRCA1 tumor suppressor gene are the most commonly detected alterations in familial breast and ovarian cancer. Although BRCA1 is required for normal mouse development, the molecular basis for its tumor suppressive function remains poorly understood. We show here that BRCA1 increases p53-dependent transcription from the p21WAF1/CIP1 and bax promoters. We also show that BRCA1 and p53 proteins interact both in vitro and in vivo. The interacting regions map, in vitro, to aa 224-500 of BRCA1 and the C-terminal domain of p53. Tumor-derived transactivation-deficient BRCA1 mutants are defective in co-activation of p53-dependent transcription and a truncation mutant of BRCA1 that retains the p53-interacting region acts as a dominant inhibitor of p53-dependent transcription. BRCA1 and p53 cooperatively induce apoptosis of cancer cells. The results indicate that BRCA1 and p53 may coordinately regulate gene expression in their role as tumor suppressors.
TL;DR: A genetic screen to identify genes whose overexpression disrupts telomeric silencing isolated 10 DOT genes, which include both components and regulators of silent chromatin, suggesting that DOT1 and DOT4 normally play important roles in gene repression.
Abstract: The ends of chromosomes in Saccharomyces cerevisiae initiate a repressive chromatin structure that spreads internally and inhibits the transcription of nearby genes, a phenomenon termed telomeric silencing. To investigate the molecular basis of this process, we carried out a genetic screen to identify genes whose overexpression disrupts telomeric silencing. We thus isolated 10 DOT genes (disruptor of telomeric silencing). Among these were genes encoding chromatin component Sir4p, DNA helicase Dna2p, ribosomal protein L32, and two proteins of unknown function, Asf1p and Ifh1p. The collection also included genes that had not previously been identified: DOT1, DOT4, DOT5, DOT6, and TLC1, which encodes the RNA template component of telomerase. With the exception of TLC1, all these genes, particularly DOT1 and DOT4, also reduced silencing at other repressed loci (HM loci and rDNA) when overexpressed. Moreover, deletion of the latter two genes weakened silencing as well, suggesting that DOT1 and DOT4 normally play important roles in gene repression. DOT1 deletion also affected telomere tract length. The function of Dot1p is not known. The sequence of Dot4p suggests that it is a ubiquitin-processing protease. Taken together, the DOT genes include both components and regulators of silent chromatin.
TL;DR: Results indicate that BC-1 cells have detectable transcription of a number of KSHV genes, particularly nonconserved genes involved in cellular signal transduction and regulation, during noninduced (latent) virus culture.
Abstract: Kaposi’s sarcoma-associated herpesvirus (KSHV) gene transcription in the BC-1 cell line (KSHV and Epstein-Barr virus coinfected) was examined by using Northern analysis with DNA probes extending across the viral genome except for a 3-kb unclonable rightmost region. Three broad classes of viral gene transcription have been identified. Class I genes, such as those encoding the v-cyclin, latency-associated nuclear antigen, and v-FLIP, are constitutively transcribed under standard growth conditions, are unaffected by tetradecanoylphorbol acetate (TPA) induction, and presumably represent latent viral transcripts. Class II genes are primarily clustered in nonconserved regions of the genome and include small polyadenylated RNAs (T0.7 and T1.1) as well as most of the virus-encoded cytokines and signal transduction genes. Class II genes are transcribed without TPA treatment but are induced to higher transcription levels by TPA treatment. Class III genes are primarily structural and replication genes that are transcribed only following TPA treatment and are presumably responsible for lytic virion production. These results indicate that BC-1 cells have detectable transcription of a number of KSHV genes, particularly nonconserved genes involved in cellular signal transduction and regulation, during noninduced (latent) virus culture.
TL;DR: Results reveal that the timing of CTD phosphorylation can account for the positive and negative functions of the two kinases and provide a model for Srb10-dependent repression of genes involved in cell type specificity, meiosis, and sugar utilization.
TL;DR: In situ hybridization analysis with probes that can detect all three transcripts shows that the RNAs are detectable in a large fraction of BCBL-1 cells prior to lytic induction and in >70% of KS spindle cells in primary KS tumors, suggesting a role for their products in viral persistence and/or KSHV-associated proliferation.
Abstract: Infection with Kaposi’s sarcoma-associated herpesvirus (KSHV) is closely associated with Kaposi’s sarcoma (KS) and primary effusion lymphoma, with viral genomes present in a latent state in the majority of tumor cells. Here we describe a cluster of latently expressed viral genes whose mRNAs are generated from a common promoter. Two mRNAs in this region encode the latency-associated nuclear antigen, the product of open reading frame 73 (ORF73). The larger RNA, of 5.8 kb, is an unspliced transcript that includes ORF72 and -71 at its 3′ end; it initiates at nucleotides (nt) 127880 to 127886 from a promoter lacking recognizable TATA elements. A less abundant mRNA, of 5.4 kb, is a variant of this transcript, in which 336 nt of 5′ noncoding information has been removed by RNA splicing. A third, more abundant RNA is generated from the same promoter region via splicing from the common splice donor at nt 127813 to an acceptor 5′ to ORF72; this transcript is the presumed mRNA for ORF72, which encodes the viral cyclin D homolog. All three RNAs are 3′ coterminal. In situ hybridization analysis with probes that can detect all three transcripts shows that the RNAs are detectable in a large fraction of BCBL-1 cells prior to lytic induction and in >70% of KS spindle cells in primary KS tumors. This confirms that these transcripts are indeed latent RNAs and suggests a role for their products in viral persistence and/or KSHV-associated proliferation.
TL;DR: It is shown that the cyclin domain of hCycT1 is necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro, as well as mediate Tat transactivation in vivo.
Abstract: HIV-1 Tat activates transcription through binding to human cyclin T1, a regulatory subunit of the TAK/P-TEFb CTD kinase complex Here we show that the cyclin domain of hCycT1 is necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro, as well as mediate Tat transactivation in vivo A Tat:TAR recognition motif (TRM) was identified at the carboxy-terminal edge of the cyclin domain, and we show that hCycT1 can interact simultaneously with Tat and CDK9 on TAR RNA in vitro Alanine-scanning mutagenesis of the hCycT1 TRM identified residues that are critical for the interaction with Tat and others that are required specifically for binding of the complex to TAR RNA Interestingly, we find that the interaction between Tat and hCycT1 requires zinc as well as essential cysteine residues in both proteins Cloning and characterization of the murine CycT1 protein revealed that it lacks a critical cysteine residue (C261) and forms a weak, zinc-independent complex with HIV-1 Tat that greatly reduces binding to TAR RNA A point mutation in mCycT1 (Y261C) restores high-affinity, zinc-dependent binding to Tat and TAR in vitro, and rescues Tat transactivation in vivo Although overexpression of hCycT1 in NIH3T3 cells strongly enhances transcription from an integrated proviral promoter, we find that this fails to overcome all blocks to productive HIV-1 infection in murine cells
TL;DR: These data are the first to identify a specific protein interaction with the BRCA1 C-terminal domain and are consistent with the model that B RCA1 functions as a transcriptional coactivator.
Abstract: The breast cancer specific tumour suppressor protein, BRCA1 (refs 1,2), activates transcription when linked with a DNA-binding domain and is a component of the RNA polymerase II (Pol II) holoenzyme. We show here that RNA helicase A (RHA) protein links BRCA1 to the holoenzyme complex. The region of BRCA1 which interacts with RHA and, thus, the holoenzyme complex, corresponds to subregions of the BRCT domain of BRCA1 (ref. 9). This interaction was shown to occur in yeast nuclei, and expression in human cells of a truncated RHA molecule which retains binding to BRCA1 inhibited transcriptional activation mediated by the BRCA1 carboxy terminus. These data are the first to identify a specific protein interaction with the BRCA1 C-terminal domain and are consistent with the model that BRCA1 functions as a transcriptional coactivator.
TL;DR: The identification of the internal ribosomal-entry site, which allows the entry of ribosomes into viral mRNA independently of the 5′ mRNA end, has been solved and it is shown that VPg can be uridylylated by the poliovirus RNA polymerase 3Dpol.
Abstract: A small protein, VPg, is covalently linked to the 5′ end of the plus-stranded poliovirus genomic RNA1,2,3. Poliovirus messenger RNA, identical in nucleotide sequence to genomic RNA, is not capped at its 5′ end by the methylated structure that is common to most eukaryotic mRNAs. These discoveries presented two problems. First, as cap structures are usually required for translation of mRNA into protein, how does this uncapped viral RNA act as a template for translation? Second, what is the function of VPg? The identification of the internal ribosomal-entry site, which allows the entry of ribosomes into viral mRNA independently of the 5′ mRNA end, has solved the first conundrum4,5,6. Here we describe the resolution of the second problem. VPg is linked to the genomic RNA through the 5′-terminal uridylic acid of the RNA. We show that VPg can be uridylylated by the poliovirus RNA polymerase 3Dpol. Uridylylated VPg can then prime the transcription of polyadenylate RNA by 3Dpol to produce VPg-linked poly(U). Initiation of transcription of the poliovirus genome from the polyadenylated 3′ end therefore depends on VPg.