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PubMed2001

Repression of glucagon gene transcription by peroxisome proliferator-activated receptor gamma through inhibition of Pax6 transcriptional activity.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is involved in glucose homeostasis and synthetic PPARgamma ligands, the thiazolidinediones, a new class of antidiabetic agents that reduce insulin resistance and, as a secondary effect, reduce hepatic glucose output. PPARgamma is highly expressed in normal human pancreatic islet alpha-cells that produce glucagon. This peptide hormone is a functional antagonist of insulin stimulating hepatic glucose output. Therefore, the effect of PPARgamma and thiazolidinediones on glucagon gene transcription was investigated. After transient transfection of a glucagon-reporter fusion gene into a glucagon-producing pancreatic islet cell line, thiazolidinediones inhibited glucagon gene transcription when PPARgamma was coexpressed. They also reduced glucagon secretion and glucagon tissue levels in primary pancreatic islets. A 5'/3'-deletion and internal mutation analysis indicated that a pancreatic islet cell-specific enhancer sequence (PISCES) motif within the proximal glucagon promoter element G1 was required for PPARgamma responsiveness. This sequence motif binds the paired domain transcription factor Pax6. When the PISCES motif within G1 was mutated into a GAL4 binding site, the expression of GAL4-Pax6 restored glucagon promoter activity and PPARgamma responsiveness. GAL4-Pax6 transcriptional activity was inhibited by PPARgamma in response to thiazolidinedione treatment also at a minimal viral promoter. These results suggest that PPARgamma in a ligand-dependent but DNA binding-independent manner inhibits Pax6 transcriptional activity, resulting in inhibition of glucagon gene transcription. These data thereby define Pax6 as a novel functional target of PPARgamma and suggest that inhibition of glucagon gene expression may be among the multiple mechanisms through which thiazolidinediones improve glycemic control in diabetic subjects.

Cell Line↗

PubMed2003

Transcriptional activation of mouse mast cell Protease-7 by activin and transforming growth factor-beta is inhibited by microphthalmia-associated transcription factor.

Previous studies have revealed that activin A and transforming growth factor-beta1 (TGF-beta1) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-beta1 in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-beta pathway, transcriptionally activated mmcp-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle, inhibited Smad3-mediated mmcp-7 transcription. MITF associated with Smad3, and the C terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF-truncated N-terminal amino acids could associate with Smad3 but did not inhibit Smad3-mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominant-negative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-beta signaling in a tissue-specific manner.

Activins↗

PubMed2006

Nhp6 is a transcriptional initiation fidelity factor for RNA polymerase III transcription in vitro and in vivo.

The binding of the RNA polymerase III (pol III) transcription factor TFIIIC to the box A intragenic promoter element of tRNA genes specifies the placement of TFIIIB on upstream-lying DNA. In turn, TFIIIB recruits pol III to the promoter and specifies transcription initiating 17-19 base pairs upstream of box A. The resolution of the pol III transcription apparatus into recombinant TFIIIB, highly purified TFIIIC, and pol III is accompanied by a loss of precision in specifying where transcription initiation occurs due to heterogeneous placement of TFIIIB. In this paper we show that Nhp6a, an abundant high mobility group B (HMGB) family, non-sequence-specific DNA-binding protein in Saccharomyces cerevisiae restores transcriptional initiation fidelity to this highly purified in vitro system. Restoration of initiation fidelity requires the presence of Nhp6a prior to TFIIIB-DNA complex formation. Chemical nuclease footprinting of TFIIIC- and TFIIIB-TFIIIC-DNA complexes reveals that Nhp6a markedly alters the TFIIIC footprint over box A and reduces the size of the TFIIIB footprint on upstream DNA sequence. Analyses of unprocessed tRNAs from yeast lacking Nhp6a and its closely related paralogue Nhp6b demonstrate that Nhp6 is required for transcriptional initiation fidelity of some but not all tRNA genes, in vivo.

Base Sequence↗

PubMed2004

Coordination of transcription factor phosphorylation and histone methylation by the P-TEFb kinase during human immunodeficiency virus type 1 transcription.

The human immunodeficiency virus type 1 (HIV-1) Tat protein recruits positive transcription elongation factor b (P-TEFb) to the transactivation response (TAR) RNA structure to facilitate formation of processive transcription elongation complexes (TECs). Here we examine the role of the Tat/TAR-specified cyclin-dependent kinase 9 (CDK9) kinase activity in regulation of HIV-1 transcription elongation and histone methylation. In HIV-1 TECs, P-TEFb phosphorylates the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) and the transcription elongation factors SPT5 and Tat-SF1 in a Tat/TAR-dependent manner. Using in vivo chromatin immunoprecipitation analysis, we demonstrate the following distinct properties of the HIV-1 transcription complexes. First, the RNAP II CTD is phosphorylated at Ser 2 and Ser 5 near the promoter and at downstream coding regions. Second, the stable association of SPT5 with the TECs is dependent upon P-TEFb kinase activity. Third, P-TEFb kinase activity is critical for the induction of methylation of histone H3 at lysine 4 and lysine 36 on HIV-1 genes. Flavopiridol, a potent P-TEFb kinase inhibitor, inhibits CTD phosphorylation, stable SPT5 binding, and histone methylation, suggesting that its potent antiviral activity is due to its ability to inhibit several critical and unique steps in HIV-1 transcription elongation.

Animals↗

PubMed1990

Activation of octamer-containing promoters by either octamer-binding transcription factor 1 (OTF-1) or OTF-2 and requirement of an additional B-cell-specific component for optimal transcription of immunoglobulin promoters.

Several distinct octamer-binding transcription factors (OTFs) interact with the sequence ATTTGCAT (the octamer motif), which acts as a transcription regulatory element for a variety of differentially controlled genes. The ubiquitous OTF-1 plays a role in expression of the cell cycle-regulated histone H2b gene as well as several other genes, while the tissue-specific OTF-2 has been implicated in the tissue-specific expression of immunoglobulin genes. In an attempt to understand the apparent transcriptional selectivity of these factors, we have investigated the physical and functional characteristics of OTF-1 purified from HeLa cells and both OTF-1 and OTF-2 purified from B cells. High-resolution footprinting and mobility shift-competition assays indicated that these factors were virtually indistinguishable in binding affinities and DNA-protein contacts on either the H2b or an immunoglobulin light-chain (kappa) promoter. In addition, each of the purified factors showed an equivalent intrinsic capacity to activate transcription from either immunoglobulin promoters (kappa and heavy chain) or the H2b promoter in OTF-depleted HeLa and B-cell extracts. However, with OTF-depleted HeLa extracts, neither factor could restore immunoglobulin gene transcription to the relatively high level observed in unfractionated B-cell extracts. Restoration of full immunoglobulin gene activity appears to require an additional B-cell regulatory component which interacts with the OTFs. The additional B-cell factor could act either by facilitating interaction of OTF activation domains with components of the general transcriptional machinery or by contributing a novel activation domain.

Base Sequence↗

PubMed2002

Transcriptional activation of human CYP17 in H295R adrenocortical cells depends on complex formation among p54(nrb)/NonO, protein-associated splicing factor, and SF-1, a complex that also participates in repression of transcription.

The first 57 bp upstream of the transcription initiation site of the human CYP17 (hCYP17) gene are essential for both basal and cAMP-dependent transcription. EMSA carried out by incubating H295R adrenocortical cell nuclear extracts with radiolabeled -57/-38 probe from the hCYP17 promoter showed the formation of three DNA-protein complexes. The fastest complex contained steroidogenic factor (SF-1) and p54(nrb)/NonO, the intermediate complex contained p54(nrb)/NonO and polypyrimidine tract-binding protein-associated splicing factor (PSF), and the slowest complex contained an SF-1/PSF/p54(nrb)/NonO complex. (Bu)(2)cAMP treatment resulted in a cAMP-inducible increase in the binding intensity of only the upper complex and also activated hCYP17 gene transcription. SF-1 coimmunoprecipitated with p54(nrb)/NonO, indicating direct interaction between these proteins. Functional assays revealed that PSF represses basal transcription. Further, the repression of hCYP17 promoter-reporter construct luciferase activity resulted from PSF interacting with the corepressor mSin3A. Trichostatin A attenuated the inhibition of basal transcription, suggesting that a histone deacetylase interacts with the SF-1/PSF/p54(nrb)/NonO/mSin3A complex. Our studies lend support to the idea that the balance between transcriptional activation and repression is essential in the control of adrenocortical steroid hormone biosynthesis.

Adrenal Cortex↗

PubMed2006

Transcription factors in asthma: are transcription factors a new target for asthma therapy?

The essential features of persistent severe asthma include structural changes in the airway wall (remodelling). It is not known whether these are the sequelae of chronic inflammation or indeed its initiators. Several transcription factors have been implicated in the inflammatory process in asthma, including the glucocorticoid receptor (GR), NFkappaB, Activator Protein-1 (AP-1), Nuclear Factor of Activated T-cells (NF-AT), cyclic AMP Response Element Binding Protein and more recently, the CCAAT/Enhancer Binding Protein (C/EBP), Peroxisome Proliferator-activated Receptor (PPAR) and the bZIP transcription factor, Nrf2. Could a pathological de-regulation of one of these transcription factors explain the broad spectrum of asthma pathology and can their modulation lead to better symptom control? Although some of the transcription factors seem to be valid targets (NFkappaB, Nrf2 or STAT6) or tools (PPARgamma, -alpha and C/EBP-alpha) for new therapeutic approaches, since many transcription factors play a central role in tissue and organ homeostasis, a longterm general suppression or overexpression, would cause severe side effects in other organs. Cell type specific application of decoy or antisense oligonucleotides for NFkappaB, Nrf2 or STAT6, or specific agonists for PPARgamma and -alpha may help to control the inflammatory response in lung epithelial cells and infiltrated immune cells, but additional, unwanted, effects on other resident cells of the lung cannot be excluded and a beneficial effect over known anti-asthma drugs has first to be proven. In order to progress with such novel therapeutic strategies, the only option seems to be to link transcription factor inhibitors/activators to a cell type specific delivery system.

Animals↗

PubMed2005

PI3-kinase and PDK-1 regulate HDAC1-mediated transcriptional repression of transcription factor NF-kappaB.

PDK-1 activates PI3-kinase/Akt signaling and regulates fundamental cellular functions, such as growth and survival. NF-kB is involved in the induction of a variety of cellular genes affecting immunity, inflammation and the resistance to apoptosis induced by some anti-cancer drugs. Even though the crucial involvement of the PI3-kinase/Akt pathway in the anti-apoptotic activation of NF-kB is well known, the exact role of PDK-1 as well as PI3-kinase/Akt in NF-kB activation is not understood. Here we demonstrate that PDK-1 plays a pivotal role in transcriptional activation of NF-kB by dissociating the transcriptional co-repressor HDAC1 from the p65 subunit of NF-kB. The association of CBP with p65 was not directly modulated by PDK-1 or by PI3-kinase. Etoposide activated NF-kB through PI3-kinase/Akt, and the transcription activation domain (TAD) of p65 was further activated by wild-type PDK-1. Overexpression of a dominant negative PDK-1 mutant decreased etoposide-induced NF-kB transcription and further down-regulated the ectopic HDAC1-mediated decrease in NF-kB transcriptional activity. Thus activation of PDK-1 relieves the HDAC1-mediated repression of NF-kB that may be related to basal as well as activated transcription by NF-kB. This effect may also explain the role of the PI3-kinase/PDK-1 pathway in the anti-apoptotic function of NF-kB associated with the chemoresistance of cancer cells.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed1991

The murine adenosine deaminase promoter requires an atypical TATA box which binds transcription factor IID and transcriptional activity is stimulated by multiple upstream Sp1 binding sites.

We have explored the template and factor requirements for in vitro transcription of the GC-rich promoter of the murine adenosine deaminase gene. The core promoter consists of an A-rich sequence (TAAAAAA) 27 base pairs upstream of the initiation site which binds transcription factor IID (TFIID) and a high affinity Sp1 binding site located 27 base pairs further upstream. Multiple upstream elements increased core promoter activity 20-fold and correspond to protected regions in DNase I footprinting assays with purified Sp1 protein. Internal deletion of the TA6 element alone eliminated transcription in spite of the presence of all other promoter elements including four Sp1 binding sites. Recombinant human TFIID supported weak basal transcription in heat-treated nuclear extracts whereas a partially purified TFIID fraction from HeLa cells reconstituted a maximal level of transcription. Inclusion of 12 base pairs immediately adjacent to the proximal Sp1 site resulted in a 5-fold boost in transcriptional activity and corresponds to a second Sp1 binding site. These results serve as a basis for further exploration of the factors involved in the developmental and selective high level tissue expression of the murine adenosine deaminase gene.

Adenosine Deaminase↗

PubMed1997

Subcellular partitioning of transcription factors during osteoblast differentiation: developmental association of the AML/CBF alpha/PEBP2 alpha-related transcription factor-NMP-2 with the nuclear matrix.

The subnuclear location of transcription factors may functionally contribute to the regulation of gene expression. Several classes of gene regulators associate with the nuclear matrix in a cell type, cell growth, or cell cycle related-manner. To understand control of nuclear matrix-transcription factor interactions during tissue development, we systematically analyzed the subnuclear partitioning of a panel of transcription factors (including NMP-1/YY-1, NMP-2/AML, AP-1, and SP-1) during osteoblast differentiation using biochemical fractionation and gel shift analyses. We show that nuclear matrix association of the tissue-specific AML transcription factor NMP-2, but not the ubiquitous transcription factor YY1, is developmentally upregulated during osteoblast differentiation. Moreover, we show that there are multiple AML isoforms in mature osteoblasts, consistent with the multiplicity of AML factors that are derived from different genes and alternatively spliced cDNAs. These AML isoforms include proteins derived from the AML-3 gene and partition between distinct subcellular compartments. We conclude that the selective partitioning of the YY1 and AML transcription factors with the nuclear matrix involves a discriminatory mechanism that targets different classes and specific isoforms of gene regulatory factors to the nuclear matrix at distinct developmental stages. Our results are consistent with a role for the nuclear matrix in regulating the expression of bone-tissue specific genes during development of the mature osteocytic phenotype.

Animals↗

PubMed1994

AML1 fusion transcripts in t(3;21) positive leukemia: evidence of molecular heterogeneity and usage of splicing sites frequently involved in the generation of normal AML1 transcripts.

The t(3;21)(q26;q22) is associated with chronic myelogenous leukemia in blast crisis (CML-BC), leukemia evolving from (therapy-related) myelodysplasia, and with leukemia following other hematopoietic proliferative diseases. Molecular cytogenetic analysis and cloning of a few t(3;21) cases indicate that the breakpoints are quite heterogeneous even within a specific clinical phenotype. Interestingly some of the (3;21) breakpoints involve the AML1 gene previously found rearranged in the t(8;21) associated with acute myelogenous leukemia. AML1 is related to the Drosophila gene runt and is the human counterpart of the gene for the alpha subunit of the nuclear polyoma enhancer binding protein (PEBP2) also known as the core binding factor (CBF). In the t(3;21) AML1 was found rearranged with EAP, a gene on chromosome 3 encoding a small ribosomal protein, as well as with EV11, another gene on chromosome 3. Here we report our study of six cases of t(3;21). By using fluorescence in situ hybridization (FISH) analysis and AML1 probes we could conclude that at least in two CML-BC cases the breakpoint occurred in the AML1 intron that is disrupted by the t(8;21). An AML1/EAP fusion transcript, different from the one described in a therapy-related myelodysplasia, was detected in both CML-BC cases. This transcript is expected to result in a predicted protein containing the AML1 nuclear binding domain with an attached stretch of 17 amino acids unrelated to the EAP small ribosomal protein. In the other t(3;21) patients we could not detect an AML1/EAP transcript or an AML1/EV11 transcript. This result suggests heterogeneity of the t(3;21) at the molecular level. The AML1 chimeric transcripts identified so far, both in the t(3;21) and in the t(8;21), diverge from the normal transcripts either after exon 5 or exon 6. Here we show that in normal AML1 transcripts different splicing events are seen to occur after AML1 exon 5 as well as exon 6.

Amino Acid Sequence↗

PubMed1989

A selection for mutants of the RNA polymerase III transcription apparatus: PCF1 stimulates transcription of tRNA and 5S RNA genes.

A genetic approach has been developed to study transcription by RNA polymerase III. A pair of Schizosaccharomyces pombe nonsense suppressor tRNA genes were arranged in tandem such that expression of the downstream (supS1) tRNA suppressor was dependent upon transcription initiated by the internal promoter of the upstream (sup9-e) gene. Dominant mutant strains of Saccharomyces cerevisiae were isolated that suppress in trans the effect of an A block promoter mutation (A19) in the sup9-e gene and restore supS1 suppressor activity. Fifteen mutant strains, eight of which were independently isolated, all have elevated steady-state levels of sup9-e A19 RNA consistent with an increase in gene transcription. Extracts of a strain carrying the dominant mutant gene, PCF1, show a general 6-fold stimulation in transcription of mutant (A19) and wild-type tRNA genes and increase 5S gene transcription 4-fold compared with extracts from a wild-type strain. A transcription factor exclusion assay was used to show that the PCF1 mutation affects two distinct stages in transcription: one prior to and one after stable complex formation; and that these effects are mediated by a component of the stable complex. Further evidence of an effect during complex assembly was obtained in a time-course experiment that showed a shortened lag phase in the PCF1 extract. The results indicate that PCF1 is either a component of the stable complex or a positive regulator of its activity.

Base Sequence↗

PubMed1993

Transcriptional slippage during the transcription initiation process at a mutant lac promoter in vivo.

A C.G to A.T transversion at position +10 of the lac promoter activates a nascent sigma 70-dependent promoter (the +10A promoter). The lac +10A promoter has two unusual properties; it programs a large family of transcripts with multiple 5' ends, and its sequence bears little resemblance to other sigma 70-dependent promoters. The 5' end of the +10A in vivo mRNA was determined to contain oligo(U) sequences of varying lengths suggesting that the true start site was at a run of three T.A base-pairs located 20 to 22 bp downstream of the lac wild-type promoter start site, and that the transcription initiation process involved a transcriptional slippage event (which resulted in multiple rU incorporation). Only mutations at or near the start site and those deletions that changed the location of the start site abolished this transcriptional slippage property of the transcription initiation process. This transcriptional slippage was also found to be promoter independent because changing the lac UV5 start site to a run of five T.A base-pairs (-1 to +4) resulted in similar transcriptional slippage. Saturated mutagenesis of the +10A promoter identified a potential -10-like region and indicated that sequences immediately upstream of the -10 region contributed to the promoter's activity. Decreasing the weak -35 region homology did not change promoter strength; however, introduction of the consensus -35 hexamer TTGACA increased expression tenfold. RNA polymerase bound to the +10A promoter partially protects a 20 base-pair sequence from DNase I digestion upstream of the start site. These results suggest that RNA polymerase interacts with the +10A promoter in a different manner from that for the majority of sigma 70 promoters.

Base Sequence↗

PubMed1996

Incorrect base insertion and prematurely terminated transcripts during T7 RNA polymerase transcription elongation past benzo[a]pyrenediol epoxide-modified DNA.

DNA replication and transcription are affected adversely by the presence of bulky adducts that are generated by the covalent binding of a variety of metabolically activated environmental pollutants to cellular DNA. When these lesions are not cleared by cellular repair enzymes prior to replication, mutations and ultimately tumor initiation can occur. Transcription and DNA repair appear to be intimately connected, since certain adducts are more efficiently removed from the transcribed strands of active loci than from non-transcribed strands and other quiescent domains in the genome. The mechanism by which RNA polymerases deal with bulky adducts during DNA transcription is therefore of great interest. The availability of site-specifically modified and stereochemically defined oligodeoxyribonucleotides derived from the covalent reaction of 7r, 8t-dihydroxy-9, 10t-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with guanine residues prompted us to study the efficiencies of transcription past these lesions using bacteriophage T7 RNA polymerase. We show here that T7 RNA polymerase can bypass such lesions in a DNA template, providing that a cytosine residue is incorporated opposite anti-BPDE-modified guanine. However, when an incorrect base (most frequently a purine) is inserted opposite the modified site, the RNA polymerase stalls, and the complex dissociates, resulting in a truncated transcript. The ability of the T7 RNA polymerase to discriminate between a correct and an incorrect inserted base and, accordingly, to continue or terminate transcription, might constitute an important mechanism that ensures the fidelity of transcription past a modified base present on the transcribed strand of the DNA template.

Bacteriophage T7↗

PubMed1987

Human RNA polymerase II can prematurely terminate transcription of the adenovirus type 2 late transcription unit at a precise site that resembles a prokaryotic termination signal.

Premature termination of transcription has been demonstrated by eukaryotic RNA polymerase II at specific sites in the major late transcriptional unit of SV40 and in one of the transcriptional units of the parvovirus, minute virus of mice (MVM) (Y. Aloni and N. Hay, CRC Critical Reviews of Biochem., 18:327-383, 1985). In both cases the prematurely terminated (attenuated) RNA can be folded into a hairpin structure followed by U-residues that resemble a termination signal in prokaryotes. The experiments presented herein demonstrate premature termination of transcription 185 nucleotides (nt) downstream from the major late promoter of adenovirus type 2 (Ad2) in vivo, and in vitro in isolated nuclei and in HeLa whole cell extract. As in SV40 and MVM the attenuated RNA of Ad2 can be folded into a hairpin structure followed by U-residues. Transcription-termination was significantly reduced when ITP replaced GTP and when Br-UTP replaced UTP in the transcription reaction mixture, indicating that RNA secondary structure and the rU-dA interactions, respectively, are parts of the termination signal. Moreover, in isolated nuclei transcription-termination at the attenuation site occurred when the reaction mixture contained between 50-150 mM NaCl but not when it contained 300 mM NaCl. These results indicate that, at least in isolated nuclei, attenuation can be regulated. The possible involvement of termination factor(s) in the regulation of attenuation is discussed.

Adenoviridae↗

PubMed1991

Characterization of transcription initiation sites on the soybean mitochondrial genome allows identification of a transcription-associated sequence motif.

Transcription initiation sites on the soybean mitochondrial genome have been characterized by sequence analysis of in vitro-capped soybean mtRNAs and corresponding mtDNA regions. The most abundant, discrete soybean mtRNA species labeled by guanylyltransferase and [alpha-32P]GTP are shown to correspond to the major transcript of the atp9 gene and to a group of small RNAs consisting of a discrete 80 nucleotide (nt) species plus heterogeneous species ranging in size from 133 to 148 nt. The 133-148 nt RNAs represent a set of transcripts with a common 5' terminus and ragged 3' ends, while the 80 nt RNA corresponds to positions 53-133 of the 133 nt species. The major, discrete in vitro-capped RNA species thus correspond to primary transcripts originating at three sites located in two regions of the soybean mitochondrial genome. The sequences extending from 13 nucleotides upstream to 8 nucleotides downstream of the initiation sites for the atp9 and 133-148 nt transcripts are identical at 18 of 21 positions. Sequences closely resembling this motif are located at some other 5' transcript termini of dicot plant mitochondria. Less closely related sequences are found at transcription initiation sites of wheat and maize mitochondria.

Base Sequence↗

PubMed1974

Transcription of bacteriophage mu. An analysis of the transcription pattern in the early phase of phage development.

It has previously been shown that the transcription of Mu is asymmetric and takes place on the heavy DNA strand (Bade, 1972; Wijffelman et al., 1974). The direction of transcription of Mu has now been determined by RNA-DNA hybridizations between purified Mu-RNA and the separated strands of lambda-Mu hybrid phages. The direction of transcription is from the c-gene (immunity gene) end of the heavy strand to the beta-end (immunity distal end) (Fig. 1). Thermo-inducible, defective Mu lysogens, in which the prophage is deleted from the beta-end, have a normal early transcription pattern, but the increase of RNA at later times is absent. A defective lysogen, which contains only the immunity gene c and the genes A and B, still has an early transcription pattern similar to that of the wild-type. Therefore, we conclude that the early RNA is transcribed from that region of the Mu genome. The early Mu-RNA synthesis is negatively regulated with a minimum rate of transcription at 9 minutes after induction. Before the onset of the late RNA synthesis, at about 22 minutes there is a rather long period in which the rate of Mu-RNA synthesis slowly increases. Using DNA strands of lambda-Mu hybrids which contain only that part of the Mu-DNA on which the early RNA synthesis takes place, we have determined that during the first half in the intermediate phase only early genes are transcribed. The amount of Mu-RNA synthesized by a Mu prophage carrying the X-mutation, which influences the excision of Mu, is greatly reduced. Negative regulation of early transcription occurs normally in this mutant.

Coliphages↗

PubMed1993

Transcriptional analysis of the Chlamydia trachomatis plasmid pCT identifies temporally regulated transcripts, anti-sense RNA and sigma 70-selected promoters.

We analysed transcription of the DNA region immediately downstream of the origin of replication in the chlamydial plasmid pCT. This region comprises two convergent open reading frames (ORF7, ORF8), encoding putative polypeptides that are homologous to each other and with C-terminal domains typical of the phage integrase family of proteins. Northern blot and RNA 5' end mapping analyses indicated that both ORFs were transcribed in the late phase of the chlamydial replicative cycle. RNA mapping showed the presence of a transcript starting 31 nucleotides (nt) before the ATG start codon of ORF7, and two temporally regulated transcripts starting 59 and 89 nt upstream of the ATG start codon of ORF8. Two abundant RNA species of 225 and 415 nt were also identified as overlapping anti-sense transcripts (AS-RNAs), complementary to the 3' end of ORF8 mRNA, with identical 5' ends but different 3' ends. In vitro and in vivo experiments in Escherichia coli showed that the sigma 70-RNA polymerase complex was capable of initiating RNA synthesis at the same sites as observed in Chlamydia trachomatis for ORF7 and AS-RNA transcripts, but was not able to transcribe ORF8. In accord with this, sequences at -10 and -35 nt upstream of the RNA 5' ends resemble sigma 70 consensus promoters in the case of ORF7 and AS, but not in the case of the two ORF8 transcripts. Therefore, transcription of ORF7 and ORF8 is controlled by different types of promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗