Coupled Transcription Translation Research Articles

Développement d'un test colorimétrique pour tester la sensibilité de l'ADN polymérase du cytomégalovirus humain au foscarnet

We described a colorimetric method to determine the biochemical phenotype of wild-type and mutated cytomegalovirus (HCMV) DNA polymerases by measuring the incorporation of digoxigenin-labelled nucleotides into the growing DNA chain. Mutations V715M and E756K, which are known to confer foscarnet-resistance, were used as controls. Mutation N495K and a combination of changes K415R and S291P, both observed in foscarnet-resistant isolates, were studied. The mutations were introduced by site-directed mutagenesis into wild-type gene UL54 cloned in an expression vector and then polymerases were synthesised by using a commercially available coupled transcription–translation system. The polymerase activity was measured with and without foscarnet. The activity of polymerases containing the V715M or E756K mutations was inhibited by foscarnet at concentrations 70- and 30-fold higher than that of wild-type polymerase, respectively. Change N495K and combination of K415R and S291P, induced a five- and ten-fold decrease in susceptibility to foscarnet, respectively. The results of this non-radioactive assay were consistent with those obtained with the conventional radioactive assay. Therefore, this novel phenotypic method could be useful for the characterisation of mutations that confer HCMV resistance to foscarnet.

Rearrangements in Thyroid Hormone Receptor Charge Clusters That StabilizeBound 3,5′,5-Triiodo-L-thyronine and Inhibit HomodimerFormation

In this study, we investigated how thyroid hormone (3,5',5-triiodo-l-thyronine, T3) inhibits binding of thyroid hormone receptor (TR) homodimers, but not TR-retinoid X receptor heterodimers, to thyroid hormone response elements. Specifically we asked why a small subset of TRbeta mutations that arise in resistance to thyroid hormone syndrome inhibit both T3 binding and formation of TRbeta homodimers on thyroid hormone response elements. We reasoned that these mutations may affect structural elements involved in the coupling of T3 binding to inhibition of TR DNA binding activity. Analysis of TR x-ray structures revealed that each of these resistance to thyroid hormone syndrome mutations affects a cluster of charged amino acids with potential for ionic bond formation between oppositely charged partners. Two clusters (1 and 2) are adjacent to the dimer surface at the junction of helices 10 and 11. Targeted mutagenesis of residues in Cluster 1 (Arg338, Lys342, Asp351, and Asp355) and Cluster 2 (Arg429, Arg383, and Glu311) confirmed that the clusters are required for stable T3 binding and for optimal TR homodimer formation on DNA but also revealed that different arrangements of charged residues are needed for these effects. We propose that the charge clusters are homodimer-specific extensions of the dimer surface and further that T3 binding promotes specific rearrangements of these surfaces that simultaneously block homodimer formation on DNA and stabilize the bound hormone. Our data yield insight into the way that T3 regulates TR DNA binding activity and also highlight hitherto unsuspected T3-dependent conformational changes in the receptor ligand binding domain.

A newly identified interaction between IVa2 and pVIII proteins during porcine adenovirus type 3 infection

The adenovirus IVa2 is an intermediate viral gene product that appears to perform multiple essential roles in viral infection. Using IVa2 as bait in the yeast two-hybrid system, we screened selected open reading frames (ORFs) of porcine adenovirus (PAdV)-3 for potential interaction with IVa2. Interestingly, pVIII showed specific interaction with IVa2. The yeast two-hybrid findings were validated by GST pull-down assays, in vitro binding studies employing cell-free coupled transcription–translation products and in vitro co-immunoprecipitations using protein-specific antibodies. Finally, we demonstrated that IVa2 specifically interacts with pVIII during PAdV-3 infection.

D‐TMPP: A novel androgen‐regulated gene preferentially expressed in prostate and prostate cancer that is the first characterized member of an eukaryotic gene family

The understanding of the molecular biology of the prostate and the process of prostate carcinogenesis is brought forward by the identification and characterization of new genes specifically expressed in prostate tissue. The encoded proteins may, in addition, provide novel diagnostic and therapeutic tools in prostate carcinoma (PCa). Here, we identify the novel gene Dresden-transmembrane protein of the prostate (D-TMPP) that is overexpressed in human prostate and prostate cancer. Proceeding from a prostate-specific expressed sequence tag identified with an Affymetrix chip-based expression database, the full-length cDNA of the novel gene was isolated from prostate tissue. The potential protein-coding function of the open reading frame (ORF) was tested by in vitro transcription-coupled translation and recombinant expression in transfected prostate cancer cells. The expression pattern of D-TMPP in malignant and nonmalignant tissues and tumor cell lines was analyzed by hybridization of a radioactively labeled cDNA probe with a multiple tissue expression array and by a quantitative real-time PCR assay. The D-TMPP-mRNA encodes a putative seven-span transmembrane protein of 883 amino acids and is selectively overexpressed in prostate tissue. D-TMPP represents the first cloned and characterized transcript of a family of eukaryotic genes. D-TMPP transcripts were detected in all analyzed pairs (n = 25) of malignant and nonmalignant prostate tissues. In the androgen-dependent PCa cell line LNCaP, D-TMPP was upregulated by methyltrienolone. We describe the novel prostate-restricted molecule D-TMPP widely expressed in prostate cancer tissues.

Tetrahydrobiopterin protects phenylalanine hydroxylase activity in vivo: Implications for tetrahydrobiopterin-responsive hyperphenylalaninemia

The natural cofactor of phenylalanine hydroxylase (PAH), tetrahydrobiopterin (BH 4), regulates the enzyme activity as well as being essential in catalysis. BH 4-responsive PAH deficiency is a variant of hyperphenylalaninemia or phenylketonuria (PKU) caused by mutations in the human PAH gene that respond to oral BH 4 loading by stimulating enzyme activity and therefore lowering serum phenylalanine. Here, we showed in a coupled transcription–translation in vitro assay that upon expression in the presence of BH 4, wild-type PAH enzyme activity was enhanced. We then investigated the effect of BH 4 on PAH activity in transgenic mice that had a complete or partial deficiency in the endogenous cofactor biosynthesis. The rate of hepatic PAH enzyme activity increased significantly with BH 4 content without affecting gene expression or Pah-mRNA stability. These results indicate that BH 4 has a chaperon-like effect on PAH synthesis and/or is a protecting cofactor against enzyme auto-inactivation and degradation also in vivo. Our findings thus contribute to the understanding of the regulation of PAH by its cofactor BH 4 on an additional level and provide a molecular explanation for cofactor-responsive PKU.

A cell-free biosensor for the detection of transcriptional inducers using firefly luciferase as a reporter

A cell-free biosensor for the detection of transcription induction by specific small-molecule ligands is presented. As model systems, tetracycline and mercury-inducible promoters were used containing firefly luciferase as reporter gene. Escherichia coli S30 extract was prepared and used for coupled transcription–translation reactions. By using purified Tet repressor and MerR regulatory proteins, we could study repressor–operator interactions for optimizing the relative concentrations of each component. Previously, detection of tetracycline and mercury using similar transcriptional regulation in whole living cells has been carried out. As compared to whole-cell biosensors, our results showed better sensitivity for the detection of tetracycline and the toxic effect of mercury was avoided in the cell-free system. Also, as the system omits cell cultivation and bacterial membranes as molecule passage inhibitors, it is possible to carry out assays in much shorter times and without the use of genetically modified organisms.

Epidermal growth factor receptor stimulation activates the RNA binding protein CUG-BP1 and increases expression of C/EBPbeta-LIP in mammary epithelial cells.

The transcription factor CCAAT/enhancer binding protein beta (C/EBP beta) is a key regulator of growth and differentiation in many tissues. C/EBP beta is expressed as several distinct protein isoforms (LAP1, LAP2, and LIP) whose expression is regulated by alternative translational initiation at downstream AUG start sites. The dominant-negative LIP isoform is predominantly expressed during proliferative cellular responses and is associated with aggressive tumors. In this study, we investigated a mechanism by which the LIP isoform is translationally regulated in mammary epithelial cells. We have demonstrated that LIP expression is increased in response to activation of the epidermal growth factor receptor (EGFR) signaling pathway and that the increased expression of LIP is regulated in part by an RNA binding protein referred to as CUG repeat binding protein (CUG-BP1). Our data demonstrate that EGFR signaling results in the phosphorylation of CUG-BP1 and this leads to an increase in the binding of CUG-BP1 to C/EBP beta mRNA and elevated expression of the LIP isoform. Phosphorylation is necessary for the binding activity of CUG-BP1 and the consequent increase in LIP expression, as determined by binding assays and a cell free, transcription-coupled translation system. CUG-BP1 is thus a previously unidentified downstream target of EGFR signaling and represents a new translational regulator of LIP expression in human mammary epithelial cells.

Molecular characterization of Sp serotype strains of infectious pancreatic necrosis virus exhibiting differences in virulence

Infectious pancreatic necrosis virus (IPNV), a prototype virus of the family Birnaviridae, exhibits a high degree of antigenic variability, pathogenicity and virulence in salmonid species. The Genomic Segment A encodes all the structural (VP2 and VP3) and nonstructural (NS) proteins, whereas Segment B encodes the viral RNA-dependent RNA polymerase (VP1). We tested 3 different IPNV isolates (Sp103, Sp116 and Sp122) isolated during field outbreaks in Norway for their ability to cause mortality in fry and post-smolt of Atlantic salmon Salmo salar L. The cumulative mortality following experimental challenge in fry was 29% for Sp122 followed by 19% for Sp116 and 15% for Sp103. In post-smolt, the corresponding mortality rates were 79, 46 and 16%, respectively. Comparisons of the deduced amino acid sequences of Segments A and B of all 3 Sp strains revealed substitutions of residues in 13 positions, of which 6 are in VP2, 2 in VP3, and 5 in VP1. Our results suggest that these residues, especially those in the outer capsid VP2, may be involved in the virulence of IPNV. Genome Segment A of the Sp serotype is 3097 nucleotides long and contains a major open reading frame (ORF) encoding a polyprotein of 972 amino acids, which initiates at the second in-frame start codon at Position 119. This was ascertained by making mutants of Segment A clone using site-directed mutagenesis, followed by in vitro transcription-coupled translation reaction and immunoprecipitation analyses. In addition, Segment A also encodes a 15 kDa arginine-rich non-structural protein from a small ORF, preceding and partially overlapping the polyprotein ORF, which is truncated to 12 kDa in the virulent Sp122 strain. Moreover, Segment A could encode a novel, putative 25 kDa protein from another ORF between VP2 and VP4 coding regions, which is only detected in the Sp serotype. Segment B is 2777 nucleotides long and encodes in a single large ORF (a polypeptide of 844 amino acid residues), VP1.

Biochemical Isolation and Analysis of a Nuclear Receptor Corepressor Complex

Publisher Summary This chapter describes the initial isolation and characterization of a core Silencing Mediator of Retinoid and Thyroid Receptors (SMRT) corepressor complex from HeLa cells. The isolation of native SMRT results in the copurification of the WD40 repeat protein—TBL1 (Transducin Beta-Like protein-1) and histone deacetylase3 (HDAC3). Because many HDACs are either severely impaired or inactive when produced and purified alone, the chapter discusses a reconstitution of HDAC3 activity using a rabbit reticulocyte lysate coupled transcription–translation system. This system is used to show that SMRT/N-CoR activates HDAC3 activity without the aid of TBL1, and it can be readily adapted to screen for other potential activators of HDAC activity. The chapter also shows the ways to produce a large-scale preparation of an active SMRT–HDAC3 complex using the baculovirus infection of sf9 cells. A fundamental action of many nuclear hormone receptors is the establishment of active repression in the absence of ligand. While nuclear receptors themselves can exhibit repressive behavior via contacts with the basal transcription machinery, the corepressors N-CoR (Nuclear Receptor CoRepressor) and SMRT have emerged as the primary mediators of nuclear receptor imposed repression. Many HDACs are either inactive or have low enzymatic activity when produced recombinantly.

Protein import and processing reconstituted with isolated rat liver mitochondria and recombinant mitochondrial processing peptidase

Most mitochondrial proteins are synthesized in the cytoplasm as larger precursors carrying N-terminal matrix-targeting presequences, and are subsequently transported to the mitochondria. The presequence mediates the interaction between the precursor polypeptide and components of the mitochondrial protein import machinery, a complex apparatus that is responsible for translocation of the precursor across the two mitochondrial membranes. Once the precursor has reached the mitochondrial matrix, the presequence is removed by the general mitochondrial processing peptidase (MPP). Some precursors undergo additional processing steps carried out by specialized processing peptidases. For most mitochondrial proteins, however, cleavage by MPP is the step that precedes folding and assembly into the native form. We describe methods to isolate import-competent mitochondria from rat liver and to perform import reactions with precursor proteins synthesized in vitro by coupled transcription–translation. We also describe methods to perform in vitro processing reactions of mitochondrial precursors by recombinant MPP and to identify the cleavage sites used by this enzyme.

Molecular characterization of a novel nuclear transglutaminase that is expressed during starfish embryogenesis.

We report the constitution and molecular characterization of a novel transglutaminase (EC 2.3.2.13) that starts to accumulate specifically in the nucleus in the starfish (Asterina pectinifera) embryo after progression through the early blastula stage. The cDNA for the nuclear transglutaminase was cloned and the cDNA-deduced sequence defines a single open reading frame encoding a protein with 737 amino acids and a predicted molecular mass of 83 kDa. A comparison of this transglutaminase with other members of the gene family revealed an overall sequence identity of 33-41%. A special sequence feature of this transglutaminase, which is not found in other transglutaminases, is the presence of nuclear localization signal-like sequences in the N-terminal region. Microinjection of hybrid constructs that encode the N-terminal segment fused to reporter proteins into the germinal vesicle of an oocyte produced chimeric proteins by transcription-coupled translation. It was found that the N-terminal segment alone was sufficient to effect nuclear accumulation of an otherwise cytoplasmic protein. These results suggest that the nuclear accumulation of the transglutaminase may play an important role in nuclear remodeling during early starfish embryogenesis.

Ostip2, a novel oncoprotein that associates with the Rho exchange factor Ost.

The ost protooncogene encodes a guanine nucleotide exchange factor for the Rho family of small GTPases, RhoA and Cdc42. The N-terminal domain of Ost (Ost-N) appears to negatively regulate the oncogenic activity of the protein, as deletion of this domain drastically increases its transforming activity in NIH 3T3 cells. Using a yeast two-hybrid system, we identified five genes encoding proteins that can interact with Ost-N. One of them, designated OSTIP2 (Ost interacting protein 2), encoded a previously uncharacterized protein. The OSTIP2 product is highly expressed in skeletal muscle as a 1.2-kb transcript. Full-length OSTIP2 cDNA contained an ORF of 193 amino acids. Transcription-coupled translation of OSTIP2 cDNA in reticulocyte lysates revealed a protein product of 20 kDa, which corresponded to the predicted size of the protein. Bacterially expressed glutathione S-transferase (GST)-Ostip2 fusion protein efficiently associated in vitro with baculovirus-expressed Ost. Interestingly, expression of Ostip2 in NIH 3T3 cells efficiently induced foci of morphologically transformed cells. Moreover, inoculation of athymic (nude) mice with OSTIP2 transfectants strongly induced tumor formation. These results suggest that Ostip2 is a novel oncoprotein that can interact with the Rho exchange factor Ost.

SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein.

Recently it has been recognized that the signal recognition particle (SRP) of Escherichia coli represents a specific targeting device for hydrophobic inner membrane proteins. It has remained unclear, however, whether the bacterial SRP functions in concert with SecA, which is required for the translocation of secretory proteins across the inner membrane. Here, we have analyzed a hybrid protein constructed by fusing the signal anchor sequence of an SRP-dependent inner membrane protein (MtlA) to the mature part of an exclusively SecA-requiring secretory protein (OmpA). We show that the signal anchor sequence of MtlA confers the novel properties onto nascent chains of OmpA of being co-translationally recognized and targeted to SecY by SRP. Once targeted to SecY, ribosome-associated nascent chains of the hybrid protein, however, remain untranslocated unless SecA is present. These results indicate that SRP and SecA cooperate in a sequential, non-overlapping manner in the topogenesis of those membrane proteins which, in addition to a signal anchor sequence, harbor a substantial hydrophilic domain to be translocated into the periplasm.

The cAMP-specific Phosphodiesterase PDE4A5 Is Cleaved Downstream of Its SH3 Interaction Domain by Caspase-3: CONSEQUENCES FOR ALTERED INTRACELLULAR DISTRIBUTION

The unique N-terminal region of the cAMP-specific phosphodiesterase PDE4A5, which confers an ability to bind to certain protein SH3 domains, is cleaved during apoptosis in both Rat-1 fibroblasts and PC12 cells. Cleavage was abolished by the caspase-3-selective inhibitor, z-DEVD-CHO but not the caspase-1 selective inhibitor, z-YVAD-CHO. Caspase-3 treatment of PDE4A5, expressed either transiently in COS cells or generated in vitro by coupled transcription translation, generated a similar cleavage product of 100 kDa compared with the native 110-kDa PDE4A5. This product could be detected immunochemically with an antibody raised to a C-terminal PDE4A5 peptide but not an antibody raised to the N terminus of PDE4A5, indicating that caspase-3 caused N-terminal cleavage of PDE4A5. Deletion of the putative caspase-3 cleavage site, (69)DAVD(72), in PDE4A5, or generation of either the D72A or the D69A mutants, ablated the ability of caspase-3 to cause cleavage. The N-terminal truncate PDE4A5-DeltaP3 was engineered to mimic the caspase-cleaved product of PDE4A5. This showed altered catalytic activity and, unlike PDE4A5, was unable to interact with the SH3 domain of the tyrosyl kinase, LYN. Although both PDE4A5 and PDE4A5-DeltaP3 were localized at cell cortical regions (ruffles), the distinct perinuclear association noted for both PDE4A5 and LYN was not seen for PDE4A5-DeltaP3. Staurosporine-induced apoptosis caused a marked redistribution of PDE4A5 but not PDE4A8 in stably transfected Rat-1 cells. The PDE4-selective inhibitor, rolipram together with the adenylyl cyclase activator forskolin, caused a synergistic increase in the apoptosis of Rat-1 cells. Overexpression of PDE4A5 in Rat-1 cells protected against staurosporine-induced apoptosis in contrast to overexpression of PDE4A8, which potentiated apoptosis. PDE4A5 may be the sole PDE4 family member to provide a substrate for caspase-3 cleavage and this action serves to remove the SH3 binding domain that is unique to this isoform within the PDE4A family and to alter its intracellular targeting.

Functional Antagonism between Msx2 and CCAAT/Enhancer-binding Protein α in Regulating the Mouse Amelogenin Gene Expression Is Mediated by Protein-Protein Interaction

Ameloblast-specific amelogenin gene expression is spatiotemporally regulated during tooth development. In a previous study, the CCAAT/enhancer-binding protein alpha (C/EBPalpha) was identified as a transcriptional activator of the mouse amelogenin gene in a cell type-specific manner. Here, Msx2 is shown to repress the promoter activity of amelogenin-promoter reporter constructs independent of its intrinsic DNA binding activity. In transient cotransfection assays, Msx2 and C/EBPalpha antagonize each other in regulating the expression of the mouse amelogenin gene. Electrophoresis mobility shift assays demonstrate that Msx2 interferes with the binding of C/EBPalpha to its cognate site in the mouse amelogenin minimal promoter, although Msx2 itself does not bind to the same promoter fragment. Protein-protein interaction between Msx2 and C/EBPalpha is identified with co-immunoprecipitation analyses. Functional antagonism between Msx2 and C/EBPalpha is also observed on the stably transfected 2.2-kilobase mouse amelogenin promoter in ameloblast-like LS8 cells. Furthermore, the carboxyl-terminal residues 183-267 of Msx2 are required for protein-protein interaction, whereas the amino-terminal residues 2-97 of Msx2 play a less critical role. Among three family members tested (C/EBPalpha, -beta, and -gamma), Msx2 preferentially interacts with C/EBPalpha. Taken together, these data indicate that protein-protein interaction rather than competition for overlapping binding sites results in the functional antagonism between Msx2 and C/EBPalpha in regulating the mouse amelogenin gene expression.

In vitro expression of Penicillium janthinellum cellobiohydrolase I gene in a coupled transcription–translation system

An enzymatically-active fungal cellobiohydrolase I (CBH I) was first synthesized in a coupled reticulocyte lysate system lacking of glycosylation modification by the template DNACbh1 in the presence of T7 RNA polymerase. The synthesized CBH I had the expected size (57 kDa) and catalyzed the substrate of p-nitrophenyl-β-d-cellobioside (pNPC), and had no activity against carboxymethyl cellulose (CMC-Na). The Km and Vmax values of the CBH I for pNPC were 0.82 mmol and 0.067 μmol min−1 per μg enzyme, respectively. The results indicated that glycosylation may not be necessary for enzymatic activity of fungal cellulases.

In vitro coupled transcription translation: Effects of modification in lysate preparation on protein composition and biosynthesis activity

In vitro coupled transcription translation: Effects of modification in lysate preparation on protein composition and biosynthesis activity

Transcription-coupled translation control of AML1/RUNX1 is mediated by cap- and internal ribosome entry site-dependent mechanisms.

AML1/RUNX1 belongs to the runt domain transcription factors that are important regulators of hematopoiesis and osteogenesis. Expression of AML1 is regulated at the level of transcription by two promoters, distal (D) and proximal (P), that give rise to mRNAs bearing two distinct 5' untranslated regions (5'UTRs) (D-UTR and P-UTR). Here we show that these 5'UTRs act as translation regulators in vivo. AML1 mRNAs bearing the uncommonly long (1,631-bp) P-UTR are poorly translated, whereas those with the shorter (452-bp) D-UTR are readily translated. The low translational efficiency of the P-UTR is attributed to its length and the cis-acting elements along it. Transfections and in vitro assays with bicistronic constructs demonstrate that the D-UTR mediates cap-dependent translation whereas the P-UTR mediates cap-independent translation and contains a functional internal ribosome entry site (IRES). The IRES-containing bicistronic constructs are more active in hematopoietic cell lines that normally express the P-UTR-containing mRNAs. Furthermore, we show that the IRES-dependent translation increases during megakaryocytic differentiation but not during erythroid differentiation, of K562 cells. These results strongly suggest that the function of the P-UTR IRES-dependent translation in vivo is to tightly regulate the translation of AML1 mRNAs. The data show that AML1 expression is regulated through usage of alternative promoters coupled with IRES-mediated translation control. This IRES-mediated translation regulation adds an important new dimension to the fine-tuned control of AML1 expression.

Cloning of CCRL1, an orphan seven transmembrane receptor related to chemokine receptors, expressed abundantly in the heart.

In an attempt to identify novel chemokine receptor genes, cDNA expressed sequence tags (EST) were analyzed for a significant homology with mammalian chemokine receptors. The sequence from one of the selected EST clones was used to generate a full-length cDNA encoding a putative seven transmembrane receptor, CCRL1-CC chemokine receptor like 1. The full-length receptor encodes a polypeptide of 350 amino acids and has about 35% homology to the chemokine receptors CCR6 and CCR7. Northern blot analysis indicates predominant expression of about 5.0, 2.0 and 1.3kb mRNA forms in human heart tissue, while low-level expression of the 2.0 and 1.3kb forms was observed in lung, pancreas and spleen and in fetal tissues. In-situ hybridization confirmed the presence of CCRL1 mRNA in cardiac muscle cells. Similar to the chemokine receptor CCR6, CCRL1 maps to chromosome 6 and has one intron in the 5' untranslated region. Coupled transcription-translation of CCRL1 cDNA yielded a glycosylated polypeptide of about 45kDa.

The Study of Guanosine 5′-Diphosphate 3′-Diphosphate-mediated Transcription Regulation in Vitro Using a Coupled Transcription-Translation System

The effects of the "alarmone" guanosine 5'-diphosphate 3'-diphosphate (ppGpp) on regulation of the Salmonella typhimurium histindine operon and the Escherichia coli tRNA(leu) operon were analyzed in vitro using a DNA-dependent transcription-translation system, S-30. The expression of the hisG promoter is positively regulated by ppGpp, whereas that of the leuV promoter (of tRNA(1eu)) is negatively regulated by ppGpp. In an attempt to understand the global regulatory mechanism of ppGpp control, interrelationship between ppGpp-dependent activation and repression of gene expression was examined using these promoters as models. It has been traditionally supposed that the ppGpp-dependent regulation, at least for the activation, is by a passive mode of control: the activation of gene expression by ppGpp is a consequence of the repression of stable RNA gene expression in the condition of RNA polymerase limiting. To test this model, the ppGpp-dependent regulations of both an activable promoter (hisGp) and a repressible promoter (leuVp) were determined in vitro simultaneously using a mixed template setup. The rationale for this exercise was to see whether the ppGpp-dependent activation and repression are inversely correlated in the in vitro condition in which RNA polymerase is limiting. No correlation was observed. It was concluded that the ppGpp-dependent activation is independent of the repression. Moreover, it was proposed that ppGpp-dependent activation and repression are mediated by titratable factors, each of which operate independently.