Inhibits Transcription Initiation Research Articles

Transcription-driven DNA supercoiling counteracts H-NS-mediated gene silencing in bacterial chromatin

In all living cells, genomic DNA is compacted through interactions with dedicated proteins and/or the formation of plectonemic coils. In bacteria, DNA compaction is achieved dynamically, coordinated with dense and constantly changing transcriptional activity. H-NS, a major bacterial nucleoid structuring protein, is of special interest due to its interplay with RNA polymerase. H-NS:DNA nucleoprotein filaments inhibit transcription initiation by RNA polymerase. However, the discovery that genes silenced by H-NS can be activated by transcription originating from neighboring regions has suggested that elongating RNA polymerases can disassemble H-NS:DNA filaments. In this study, we present evidence that transcription-induced counter-silencing does not require transcription to reach the silenced gene; rather, it exerts its effect at a distance. Counter-silencing is suppressed by introducing a DNA gyrase binding site within the intervening segment, suggesting that the long-range effect results from transcription-driven positive DNA supercoils diffusing toward the silenced gene. We propose a model wherein H-NS:DNA complexes form in vivo on negatively supercoiled DNA, with H-NS bridging the two arms of the plectoneme. Rotational diffusion of positive supercoils generated by neighboring transcription will cause the H-NS-bound negatively-supercoiled plectoneme to “unroll” disrupting the H-NS bridges and releasing H-NS.

Expression of RNA polymerase I catalytic core is influenced by RPA12.

RNA Polymerase I (Pol I) has recently been recognized as a cancer therapeutic target. The activity of this enzyme is essential for ribosome biogenesis and is universally activated in cancers. The enzymatic activity of this multi-subunit complex resides in its catalytic core composed of RPA194, RPA135, and RPA12, a subunit with functions in RNA cleavage, transcription initiation and elongation. Here we explore whether RPA12 influences the regulation of RPA194 in human cancer cells. We use a specific small-molecule Pol I inhibitor BMH-21 that inhibits transcription initiation, elongation and ultimately activates the degradation of Pol I catalytic subunit RPA194. We show that silencing RPA12 causes alterations in the expression and localization of Pol I subunits RPA194 and RPA135. Furthermore, we find that despite these alterations not only does the Pol I core complex between RPA194 and RPA135 remain intact upon RPA12 knockdown, but the transcription of Pol I and its engagement with chromatin remain unaffected. The BMH-21-mediated degradation of RPA194 was independent of RPA12 suggesting that RPA12 affects the basal expression, but not the drug-inducible turnover of RPA194. These studies add to knowledge defining regulatory factors for the expression of this Pol I catalytic subunit.

Quasi Markov state models elucidates the role of bacterial RNA polymerase loading gate and trigger loop dynamics in antibiotics inhibition.

The rise of antibiotic resistance urges the development of new antibiotics. Bacterial RNA Polymerase (RNAP) transcribes DNA to RNA and is an effective target for antibiotics. RNAP has a crab-claw-like shape with two pincers, clamp and β-lobe domains, forming a loading gate. During the initiation of transcription,the opening of the loading gate is necessary to load the template DNA. Myx is an antibiotic that binds to the switch 2 region, under the clamp, to prevent clamp closing/opening and inhibit transcription initiation. To study the inhibition mechanism of Myx in atomic resolution, we built quasi-Markov State Models (qMSM) based on extensive molecular dynamics (MD) simulations. qMSM can obtain the slow timescale dynamics of complex conformational change using shorter MD simulations compared to traditional MSM. qMSM identifies four states of clamp opening: closed, two partially closed, and open states. We have shown that the opening of the Myx binding site only occurs in a partially closed state, which allows binding of Myx. This suggests that Myx selectively binds to a partially closed state and follows the conformational selection mechanism. Notably, we find that β-lobe opening is sufficient for loading of DNA during transcription initiation. This highlights the critical role of β-lobe to initiate transcription and β-lobe's potential as a target for future antibiotics development. We also utilize qMSMto study the folding of the trigger loop (TL) of RNAP. During RNA elongation, TL closes/folds to stabilize the NTP substrate in the active site for catalysis. CBR is antibiotics that inhibit the folding of TL. Studying the molecular mechanism of TL folding will be useful to understand the inhibition mechanism of antibiotics, CBR, and future drug design targeting TL.

Time-resolved analysis of transcription kinetics in single live mammalian cells

In eukaryotic cells, RNA polymerase II synthesizes mRNA in three stages, initiation, elongation, and termination, and numerous factors determine how quickly a gene is transcribed to produce mRNA molecules through these steps. However, there are few techniques available to measure the rate of each step in living cells, which prevents a better understanding of transcriptional regulation. Here, we present a quantitative analysis method to extract kinetic rates of transcription from time-lapse imaging data of fluorescently labeled mRNA in live cells. Using embryonic fibroblasts cultured from two knock-in mouse models, we monitored transcription of β-actin and Arc mRNA labeled with MS2 and PP7 stem–loop systems, respectively. After inhibiting transcription initiation, we measured the elongation rate and the termination time by fitting the time trace of transcription intensity with a mathematical model function. We validated our results by comparing them with those from an autocorrelation analysis and stochastic simulations. This live-cell transcription analysis method will be useful for studying the regulation of elongation and termination steps, providing insight into the diverse mechanisms of transcriptional processes.

Nuclear export restricts Gdown1 to a mitotic function.

Approximately half of purified mammalian RNA polymerase II (Pol II) is associated with a tightly interacting sub-stoichiometric subunit, Gdown1. Previous studies have established that Gdown1 inhibits transcription initiation through competitive interactions with general transcription factors and blocks the Pol II termination activity of transcription termination factor 2 (TTF2). However, the biological functions of Gdown1 remain poorly understood. Here, we utilized genetic, microscopic, and multi-omics approaches to functionally characterize Gdown1 in three human cell lines. Acute depletion of Gdown1 caused minimal direct effects on transcription. We show that Gdown1 resides predominantly in the cytoplasm of interphase cells, shuttles between the cytoplasm and nucleus, and is regulated by nuclear export. Gdown1 enters the nucleus at the onset of mitosis. Consistently, genetic ablation of Gdown1 is associated with partial de-repression of mitotic transcription, and Gdown1 KO cells present with evidence of aberrant mitoses coupled to p53 pathway activation. Evidence is presented demonstrating that Gdown1 modulates the combined functions of purified productive elongation factors PAF1C, RTF1, SPT6, DSIF and P-TEFb in vitro. Collectively, our findings support a model wherein the Pol II-regulatory function of Gdown1 occurs during mitosis and is required for genome integrity.

DNA supercoiling-mediated collective behavior of co-transcribing RNA polymerases.

Multiple RNA polymerases (RNAPs) transcribing a gene have been known to exhibit collective group behavior, causing the transcription elongation rate to increase with the rate of transcription initiation. Such behavior has long been believed to be driven by a physical interaction or ‘push’ between closely spaced RNAPs. However, recent studies have posited that RNAPs separated by longer distances may cooperate by modifying the DNA segment under transcription. Here, we present a theoretical model incorporating the mechanical coupling between RNAP translocation and the DNA torsional response. Using stochastic simulations, we demonstrate DNA supercoiling-mediated long-range cooperation between co-transcribing RNAPs. We find that inhibiting transcription initiation can slow down the already recruited RNAPs, in agreement with recent experimental observations, and predict that the average transcription elongation rate varies non-monotonically with the rate of transcription initiation. We further show that while RNAPs transcribing neighboring genes oriented in tandem can cooperate, those transcribing genes in divergent or convergent orientations can act antagonistically, and that such behavior holds over a large range of intergenic separations. Our model makes testable predictions, revealing how the mechanical interplay between RNAPs and the DNA they transcribe can govern transcriptional dynamics.

Four paralogous Gfh factors in the extremophilic bacterium Deinococcus peraridilitoris have distinct effects on various steps of transcription

Gre factors are ubiquitous transcription regulators that stimulate co-transcriptional RNA cleavage by bacterial RNA polymerase (RNAP). Here, we show that the stress-resistant bacterium Deinococcus peraridilitoris encodes four Gre factor homologs, Gfh proteins, that have distinct effects on transcription by RNAP. Two of the factors, Gfh1α and Gfh2β inhibit transcription initiation, and one of them, Gfh1α can also regulate transcription elongation. We show that this factor strongly stimulates transcriptional pausing and intrinsic termination in the presence of manganese ions but has no effect on RNA cleavage. Thus, some Gfh factors encoded by Deinococci serve as lineage-specific transcription inhibitors that may play a role in stress resistance, while the functions of others remain to be discovered.

Change in inorganic phosphate physical state can regulate transcription

ABSTRACTInorganic phosphate (Pi), a ubiquitous metabolite, is involved in all major biochemical pathways. We demonstrate that, in vitro, MgHPO4 (the intracellular Pi form) at physiological concentrations can exist in a metastable supersaturated dissolved state or as a precipitate. We have shown that in solution, MgHPO4 strongly stimulates exonuclease nascent transcript cleavage by RNA polymerase. We report here that MgHPO4 precipitate selectively and efficiently inhibits transcription initiation in vitro. In view of the MgHPO4 solubility and in vitro sensitivity of RNA synthesis to MgHPO4 precipitate, at physiological concentrations, MgHPO4 should cause a 50–98% inhibition of cellular RNA synthesis, thus exerting a strong regulatory action. The effects of Pi on transcription in vivo will, therefore, reflect the physical state of intracellular Pi.

Vesiculopolins, a New Class of Anti-Vesiculoviral Compounds, Inhibit Transcription Initiation of Vesiculoviruses

Vesicular stomatitis virus (VSV) represents a promising platform for developing oncolytic viruses, as well as vaccines against significant human pathogens. To safely control VSV infection in humans, small-molecule drugs that selectively inhibit VSV infection may be needed. Here, using a cell-based high-throughput screening assay followed by an in vitro transcription assay, compounds with a 7-hydroxy-6-methyl-3,4-dihydroquinolin-2(1H)-one structure and an aromatic group at position 4 (named vesiculopolins, VPIs) were identified as VSV RNA polymerase inhibitors. The most effective compound, VPI A, inhibited VSV-induced cytopathic effects and in vitro mRNA synthesis with micromolar to submicromolar 50% inhibitory concentrations. VPI A was found to inhibit terminal de novo initiation rather than elongation for leader RNA synthesis, but not mRNA capping, with the VSV L protein, suggesting that VPI A is targeted to the polymerase domain in the L protein. VPI A inhibited transcription of Chandipura virus, but not of human parainfluenza virus 3, suggesting that it specifically acts on vesiculoviral L proteins. These results suggest that VPIs may serve not only as molecular probes to elucidate the mechanisms of transcription of vesiculoviruses, but also as lead compounds to develop antiviral drugs against vesiculoviruses and other related rhabdoviruses.

Structural basis for transcription antitermination at bacterial intrinsic terminator

Bacteriophages typically hijack the host bacterial transcriptional machinery to regulate their own gene expression and that of the host bacteria. The structural basis for bacteriophage protein-mediated transcription regulation—in particular transcription antitermination—is largely unknown. Here we report the 3.4 Å and 4.0 Å cryo-EM structures of two bacterial transcription elongation complexes (P7-NusA-TEC and P7-TEC) comprising the bacteriophage protein P7, a master host-transcription regulator encoded by bacteriophage Xp10 of the rice pathogen Xanthomonas oryzae pv. Oryzae (Xoo) and discuss the mechanisms by which P7 modulates the host bacterial RNAP. The structures together with biochemical evidence demonstrate that P7 prevents transcription termination by plugging up the RNAP RNA-exit channel and impeding RNA-hairpin formation at the intrinsic terminator. Moreover, P7 inhibits transcription initiation by restraining RNAP-clamp motions. Our study reveals the structural basis for transcription antitermination by phage proteins and provides insights into bacterial transcription regulation.

StpA and Hha stimulate pausing by RNA polymerase by promoting DNA-DNA bridging of H-NS filaments.

In enterobacteria, AT-rich horizontally acquired genes, including virulence genes, are silenced through the actions of at least three nucleoid-associated proteins (NAPs): H-NS, StpA and Hha. These proteins form gene-silencing nucleoprotein filaments through direct DNA binding by H-NS and StpA homodimers or heterodimers. Both linear and bridged filaments, in which NAPs bind one or two DNA segments, respectively, have been observed. Hha can interact with H-NS or StpA filaments, but itself lacks a DNA-binding domain. Filaments composed of H-NS alone can inhibit transcription initiation and, in the bridged conformation, slow elongating RNA polymerase (RNAP) by promoting backtracking at pause sites. How the other NAPs modulate these effects of H-NS is unknown, despite evidence that they help regulate subsets of silenced genes in vivo (e.g. in pathogenicity islands). Here we report that Hha and StpA greatly enhance H-NS-stimulated pausing by RNAP at 20°C. StpA:H-NS or StpA-only filaments also stimulate pausing at 37°C, a temperature at which Hha:H-NS or H-NS-only filaments have much less effect. In addition, we report that both Hha and StpA greatly stimulate DNA–DNA bridging by H-NS filaments. Together, these observations indicate that Hha and StpA can affect H-NS-mediated gene regulation by stimulating bridging of H-NS/DNA filaments.

The transcript cleavage factor paralogue TFS4 is a potent RNA polymerase inhibitor

TFIIS-like transcript cleavage factors enhance the processivity and fidelity of archaeal and eukaryotic RNA polymerases. Sulfolobus solfataricus TFS1 functions as a bona fide cleavage factor, while the paralogous TFS4 evolved into a potent RNA polymerase inhibitor. TFS4 destabilises the TBP–TFB–RNAP pre-initiation complex and inhibits transcription initiation and elongation. All inhibitory activities are dependent on three lysine residues at the tip of the C-terminal zinc ribbon of TFS4; the inhibition likely involves an allosteric component and is mitigated by the basal transcription factor TFEα/β. A chimeric variant of yeast TFIIS and TFS4 inhibits RNAPII transcription, suggesting that the molecular basis of inhibition is conserved between archaea and eukaryotes. TFS4 expression in S. solfataricus is induced in response to infection with the Sulfolobus turreted icosahedral virus. Our results reveal a compelling functional diversification of cleavage factors in archaea, and provide novel insights into transcription inhibition in the context of the host–virus relationship.

Regulation of transcription initiation by Gfh factors from Deinococcus radiodurans.

Transcription factors of the Gre family bind within the secondary channel of bacterial RNA polymerase (RNAP) directly modulating its catalytic activities. Universally conserved Gre factors activate RNA cleavage by RNAP, by chelating catalytic metal ions in the RNAP active site, and facilitate both promoter escape and transcription elongation. Gfh factors are Deinococcus/Thermus-specific homologues of Gre factors whose transcription functions remain poorly understood. Recently, we found that Gfh1 and Gfh2 proteins from Deinococcus radiodurans dramatically stimulate RNAP pausing during transcription elongation in the presence of Mn2+, but not Mg2+, ions. In contrast, we show that Gfh1 and Gfh2 moderately inhibit transcription initiation in the presence of either Mg2+ or Mn2+ ions. By using a molecular beacon assay, we demonstrate that Gfh1 and Gfh2 do not significantly change promoter complex stability or the rate of promoter escape by D. radiodurans RNAP. At the same time, Gfh factors significantly increase the apparent KM value for the 5'-initiating nucleotide, without having major effects on the affinity of metal ions for the RNAP active site. Similar inhibitory effects of Gfh factors are observed for transcription initiation on promoters recognized by the principal and an alternative σ factor. In summary, our data suggest that D. radiodurans Gfh factors impair the binding of initiating substrates independently of the metal ions bound in the RNAP active site, but have only mild overall effects on transcription initiation. Thus the mechanisms of modulation of RNAP activity by these factors are different for various steps of transcription.

From indole to pyrrole, furan, thiophene and pyridine: Search for novel small molecule inhibitors of bacterial transcription initiation complex formation

The search for small molecules capable of inhibiting transcription initiation in bacteria has resulted in the synthesis of N,N′-disubstituted hydrazines and imine-carbohydrazides comprised of indole, pyridine, pyrrole, furan and thiophene using the respective trichloroacetyl derivatives, carbohydrazides and aldehydes. Replacement of the indole moiety by smaller heterocycles linked by CONHNC linkers afforded a broad variety of compounds efficiently targeting the RNA polymerase–σ70/σA interaction as determined by ELISA and exhibiting increased inhibition of the growth of Escherichia coli compared to Bacillus subtilis in culture. The structural features of the synthesized transcription initiation inhibitors needed for antibacterial activity were identified employing molecular modelling and structure–activity relationship (SAR) studies.

Aptamers to the sigma factor mimic promoter recognition and inhibit transcription initiation by bacterial RNA polymerase

Promoter recognition by bacterial RNA polymerase (RNAP) is a multi-step process involving multiple protein-DNA interactions and several structural and kinetic intermediates which remain only partially characterized. We used single-stranded DNA aptamers containing specific promoter motifs to probe the interactions of the Thermus aquaticus RNAP σA subunit with the −10 promoter element in the absence of other parts of the promoter complex. The aptamer binding decreased intrinsic fluorescence of the σ subunit, likely as a result of interactions between the −10 element and conserved tryptophan residues of the σ DNA-binding region 2. By monitoring these changes, we demonstrated that DNA binding proceeds through a single rate-limiting step resulting in formation of very stable complexes. Deletion of the N-terminal domain of the σA subunit increased the rate of aptamer binding while replacement of this domain with an unrelated N-terminal region 1.1 from the Escherichia coli σ70 subunit restored the original kinetics of σ-aptamer interactions. The results demonstrate that the key step in promoter recognition can be modelled in a simple σ-aptamer system and reveal that highly divergent N-terminal domains similarly modulate the DNA-binding properties of the σ subunit. The aptamers efficiently suppressed promoter-dependent transcription initiation by the holoenzyme of RNA polymerase, suggesting that they may be used for development of novel transcription inhibitors.

Homozygousmdm2SNP309 cancer cells with compromised transcriptional elongation at p53 target genes are sensitive to induction of p53-independent cell death

A single nucleotide polymorphism (T to G) in the mdm2 P2 promoter, mdm2 SNP309, leads to MDM2 overexpression promoting chemotherapy resistant cancers. Two mdm2 G/G SNP309 cancer cell lines, MANCA and A875, have compromised wild-type p53 that co-localizes with MDM2 on chromatin. We hypothesized that MDM2 in these cells inhibited transcription initiation at the p53 target genes p21 and puma. Surprisingly, following etoposide treatment transcription initiation occurred at the compromised target genes in MANCA and A875 cells similar to the T/T ML-1 cell line. In all cell lines tested there was equally robust recruitment of total and initiated RNA polymerase II (Pol II). We found that knockdown of MDM2 in G/G cells moderately increased expression of subsets of p53 target genes without increasing p53 stability. Importantly, etoposide and actinomycin D treatments increased histone H3K36 trimethylation in T/T, but not G/G cells, suggesting a G/G correlated inhibition of transcription elongation. We therefore tested a chemotherapeutic agent (8-amino-adenosine) that induces p53-independent cell death for higher clinically relevant cytotoxicity. We demonstrated that T/T and G/G mdm2 SNP309 cells were equally sensitive to 8-amino-adenosine induced cell death. In conclusion for cancer cells overexpressing MDM2, targeting MDM2 may be less effective than inducing p53-independent cell death.

Abstract PR02: Specific inhibition of hTERT expression in melanoma by targeting common promoter mutations which cause quadruplex DNA instability

Abstract Background: Telomerase reverse transcriptase (hTERT) is a catalytic subunit of the enzyme telomerase. It has recently been shown that the hTERT promoter is commonly mutated (>75% of cases) in malignant melanoma. These mutations occur at four sites in a G-rich region of the promoter which has previously been shown to form quadruplex DNA and to downregulate hTERT expression. We have tested the hypothesis that mutations in the quadruplex-forming region of the hTERT promoter destabilize quadruplex-formation resulting in increased hTERT expression and cellular proliferation, thus providing a novel therapeutic target for melanoma. Materials and Methods: Quadruplex formation by the mutated and wild type hTERT promoter oligonucleotides was determined by circular dichroism. Analytical ultracentrifugation was used for sedimentation equilibrium analysis. Thermal denaturation was used to characterize the relative stability of the mutated and wild type oligonucleotides. The growth inhibitory effect of mutated and wild type oligonucleotides were determined in four cell lines which contained mutated or wild type hTERT promoter sequences. Cell proliferation was characterized by MTT and cell counting. Results: In order to characterize the effects of the hTERT mutations, the biophysical properties of structures formed by wild-type and mutant TERT sequences were explored by several methods. Circular dichroism and thermal denaturation studies showed that all sequences formed quadruplex structures but that those formed by the mutated sequences were markedly less stable than the wild-type. Analytical ultracentrifugation showed that all sequences formed one major unimolecular folded form but that mutant sequences had a greater tendency to form misfolded aggregated species than the wild-type. Addition of the quadruplex binder TmPyP4 to the mutant sequences lessened that amount of such aggregates and resulted in sedimentation profiles that closely resembled the wild-type sequences. Treatment of cells with mutated hTERT promoter sequence with oligonucleotides encoding the mutated or wild type sequence resulted in significant growth inhibition that was time and concentration dependent. DNA crosslinking studies indicate that the G-rich oligonucleotide is binding to the C-rich strand by Watson-Crick base pairing suggesting that it inhibits transcription initiation by strand invasion. A similar growth inhibitory effect was seen in cells exposed to quadruplex stabilizing agents. Conclusions: The common mutations in the hTERT promoter destabilize quadruplex formation and likely prevent quadruplex-mediated transcriptional silencing. This instability can be overcome by quadruplex-binding drugs and/or oligonucleotides encoding this sequence. The growth of cell lines containing the hTERT promoter mutations is inhibited by oligonucleotides encoding the wild type and mutated sequences. These oligonucleotides stabilize the mutated genomic quadruplex structure, resulting in transcriptional silencing. This approach has obvious clinical potential. This abstract is also being presented as Poster A07. Citation Format: Donald M. Miller, Alexandra Sokolova, Francine Rezzoug, Shelia Thomas, Jonathan Chaires, Jonathan Chaires, William Dean, John Trent. Specific inhibition of hTERT expression in melanoma by targeting common promoter mutations which cause quadruplex DNA instability. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr PR02.

Respiratory Syncytial Virus Inhibitor AZ-27 Differentially Inhibits Different Polymerase Activities at the Promoter.

Respiratory syncytial virus (RSV) is the leading cause of pediatric respiratory disease. RSV has an RNA-dependent RNA polymerase that transcribes and replicates the viral negative-sense RNA genome. The large polymerase subunit (L) has multiple enzymatic activities, having the capability to synthesize RNA and add and methylate a cap on each of the viral mRNAs. Previous studies (H. Xiong et al., Bioorg Med Chem Lett, 23:6789-6793, 2013, http://dx.doi.org/10.1016/j.bmcl.2013.10.018; C. L. Tiong-Yip et al., Antimicrob Agents Chemother, 58:3867-3873, 2014, http://dx.doi.org/10.1128/AAC.02540-14) had identified a small-molecule inhibitor, AZ-27, that targets the L protein. In this study, we examined the effect of AZ-27 on different aspects of RSV polymerase activity. AZ-27 was found to inhibit equally both mRNA transcription and genome replication in cell-based minigenome assays, indicating that it inhibits a step common to both of these RNA synthesis processes. Analysis in an in vitro transcription run-on assay, containing RSV nucleocapsids, showed that AZ-27 inhibits synthesis of transcripts from the 3' end of the genome to a greater extent than those from the 5' end, indicating that it inhibits transcription initiation. Consistent with this finding, experiments that assayed polymerase activity on the promoter showed that AZ-27 inhibited transcription and replication initiation. The RSV polymerase also can utilize the promoter sequence to perform a back-priming reaction. Interestingly, addition of AZ-27 had no effect on the addition of up to three nucleotides by back-priming but inhibited further extension of the back-primed RNA. These data provide new information regarding the mechanism of inhibition by AZ-27. They also suggest that the RSV polymerase adopts different conformations to perform its different activities at the promoter. Currently, there are no effective antiviral drugs to treat RSV infection. The RSV polymerase is an attractive target for drug development, but this large enzymatic complex is poorly characterized, hampering drug development efforts. AZ-27 is a small-molecule inhibitor previously shown to target the RSV large polymerase subunit (C. L. Tiong-Yip et al., Antimicrob Agents Chemother, 58:3867-3873, 2014, http://dx.doi.org/10.1128/AAC.02540-14), but its inhibitory mechanism was unknown. Understanding this would be valuable both for characterizing the polymerase and for further development of inhibitors. Here, we show that AZ-27 inhibits an early stage in mRNA transcription, as well as genome replication, by inhibiting initiation of RNA synthesis from the promoter. However, the compound does not inhibit back priming, another RNA synthesis activity of the RSV polymerase. These findings provide insight into the different activities of the RSV polymerase and will aid further development of antiviral agents against RSV.

Regulation of ribosomal RNA synthesis in T cells: requirement for GTP and Ebp1

AbstractMycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, an effective immunosuppressive drug. Both MPA and mycophenolate mofetil are highly specific inhibitors of guanine nucleotide synthesis and of T-cell activation. However, the mechanism by which guanine nucleotide depletion suppresses T-cell activation is unknown. Depletion of GTP inhibits ribosomal RNA synthesis in T cells by inhibiting transcription initiation factor I (TIF-IA), a GTP-binding protein that recruits RNA polymerase I to the ribosomal DNA promoter. TIF-IA–GTP binds the ErbB3-binding protein 1, and together they enhance the transcription of proliferating cell nuclear antigen (PCNA). GTP binding by TIF-IA and ErbB3-binding protein 1 phosphorylation by protein kinase C δ are both required for optimal PCNA expression. The protein kinase C inhibitor sotrastaurin markedly potentiates the inhibition of ribosomal RNA synthesis, PCNA expression, and T-cell activation induced by MPA, suggesting that the combination of the two agents are more highly effective than either alone in inducing immunosuppression.

CcpA-Mediated Catabolite Activation of the Bacillus subtilis ilv-leu Operon and Its Negation by Either CodY- or TnrA-Mediated Negative Regulation

The Bacillus subtilis ilv-leu operon functions in the biosynthesis of branched-chain amino acids. It undergoes catabolite activation involving a promoter-proximal cre which is mediated by the complex of CcpA and P-Ser-HPr. This activation of ilv-leu expression is negatively regulated through CodY binding to a high-affinity site in the promoter region under amino acid-rich growth conditions, and it is negatively regulated through TnrA binding to the TnrA box under nitrogen-limited growth conditions. The CcpA-mediated catabolite activation of ilv-leu required a helix face-dependent interaction of the complex of CcpA and P-Ser-HPr with RNA polymerase and needed a 19-nucleotide region upstream of cre for full activation. DNase I footprinting indicated that CodY binding to the high-affinity site competitively prevented the binding of the complex of CcpA and P-Ser-HPr to cre. This CodY binding not only negated catabolite activation but also likely inhibited transcription initiation from the ilv-leu promoter. The footprinting also indicated that TnrA and the complex of CcpA and P-Ser-HPr simultaneously bound to the TnrA box and the cre site, respectively, which are 112 nucleotides apart; TnrA binding to its box was likely to induce DNA bending. This implied that interaction of TnrA bound to its box with the complex of CcpA and P-Ser-HPr bound to cre might negate catabolite activation, but TnrA bound to its box did not inhibit transcription initiation from the ilv-leu promoter. Moreover, this negation of catabolite activation by TnrA required a 26-nucleotide region downstream of the TnrA box.