Cell-free Transcription Assay Research Articles

Design of synthetic promoters for cyanobacteria with generative deep-learning model.

Deep generative models, which can approximate complex data distribution from large datasets, are widely used in biological dataset analysis. In particular, they can identify and unravel hidden traits encoded within a complicated nucleotide sequence, allowing us to design genetic parts with accuracy. Here, we provide a deep-learning based generic framework to design and evaluate synthetic promoters for cyanobacteria using generative models, which was in turn validated with cell-free transcription assay. We developed a deep generative model and a predictive model using a variational autoencoder and convolutional neural network, respectively. Using native promoter sequences of the model unicellular cyanobacterium Synechocystis sp. PCC 6803 as a training dataset, we generated 10000 synthetic promoter sequences and predicted their strengths. By position weight matrix and k-mer analyses, we confirmed that our model captured a valid feature of cyanobacteria promoters from the dataset. Furthermore, critical subregion identification analysis consistently revealed the importance of the -10 box sequence motif in cyanobacteria promoters. Moreover, we validated that the generated promoter sequence can efficiently drive transcription via cell-free transcription assay. This approach, combining in silico and in vitro studies, will provide a foundation for the rapid design and validation of synthetic promoters, especially for non-model organisms.

Enhancer–promoter entanglement explains their transcriptional interdependence

Enhancers not only activate target promoters to stimulate messenger RNA (mRNA) synthesis, but they themselves also undergo transcription to produce enhancer RNAs (eRNAs), the significance of which is not well understood. Transcription at the participating enhancer-promoter pair appears coordinated, but it is unclear why and how. Here, we employ cell-free transcription assays using constructs derived from the human GREB1 locus to demonstrate that transcription at an enhancer and its target promoter is interdependent. This interdependence is observable under conditions where direct enhancer-promoter contact (EPC) takes place. We demonstrate that transcription activation at a participating enhancer-promoter pair is dependent on i) the mutual availability of the enhancer and promoter, ii) the state of transcription at both the enhancer and promoter, iii) local abundance of both eRNA and mRNA, and iv) direct EPC. Our results suggest transcriptional interdependence between the enhancer and the promoter as the basis of their transcriptional concurrence and coordination throughout the genome. We propose a model where transcriptional concurrence, coordination and interdependence are possible if the participating enhancer and promoter are entangled in the form of EPC, reside in a proteinaceous bubble, and utilize shared transcriptional resources and regulatory inputs.

Chromatin Kinases Act on Transcription Factors and Histone Tails in Regulation of Inducible Transcription

The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate the connections between inflammatory signaling pathways and chromatin through genomic footprinting of kinase activity and unbiased identification of prominent histone phosphorylation events.We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen- and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.

Targeting chromatin kinases reveals their role in inducible transcription of inflammatory genes

Abstract The inflammatory response requires selective, rapid regulation of chromatin and induction of transcription for defense against pathogens and environmental insult, and also features abundant kinase activity. We sought to identify the connections between kinase pathway activity and regulation of chromatin through unbiased identification and genomic foot printing of prominent histone phosphorylation events. We identified histone H3 serine 28 phosphorylation (H3S28p) as the principal stimulation-dependent histone modification with enrichment at induced inflammatory genes in mouse macrophages stimulated with bacterial lipopolysaccharide (LPS). Use of chemical inhibitors and genetic rescue identified mitogen- and stress-activated protein kinases (MSK) as the primary mediators of H3S28p in macrophages. Cell-free transcription assays demonstrated that H3S28p directly promotes transcription. Further, we show that MSK can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Indeed, inhibition of MSK in macrophages selectively reduced transcription of stimulation-induced genes. Our results indicate MSK as a nexus of signaling inputs controlling multiple downstream regulators of inducible transcription.

Reverse Transcriptase-Coupled Quantitative Real Time PCR Analysis of Cell-Free Transcription on the Chromatin-Assembled p21 Promoter

BackgroundCell-free eukaryotic transcription assays have contributed tremendously to the current understanding of the molecular mechanisms that govern transcription at eukaryotic promoters. Currently, the conventional G-less cassette transcription assay is one of the simplest and fastest methods for measuring transcription in vitro. This method requires several components, including the radioisotope labelling of RNA product during the transcription reaction followed by visualization of transcripts using autoradiography.Methodology/Principal FindingsTo further simplify and expedite the conventional G-less cassette transcription assay, we have developed a method to incorporate a reverse transcriptase-coupled quantitative real time PCR (RT-qPCR). By using DNA template depletion steps that include DNA template immobilization, Trizol extraction and DNase I treatment, we have successfully enriched p21 promoter-driven transcripts over DNA templates. The quantification results of RNA transcripts using the RT-qPCR assay were comparable to the results of the conventional G-less cassette transcription assay both in naked DNA and chromatin-assembled templates.ConclusionsWe first report a proof-of-concept demonstration that incorporating RT-qPCR in cell-free transcription assays can be a simpler and faster alternative method to the conventional radioisotope-mediated transcription assays. This method will be useful for developing high throughput in vitro transcription assays and provide quantitative data for RNA transcripts generated in a defined cell-free transcription reaction.

Functional Analysis of a Novelcis-Acting Regulatory Region within the Human Ankyrin Gene (ANK-1) Promoter

The characterization of atypical mutations in loci associated with diseases is a powerful tool to discover novel regulatory elements. We previously identified a dinucleotide deletion in the human ankyrin-1 gene (ANK-1) promoter that underlies ankyrin-deficient hereditary spherocytosis. The presence of the deletion was associated with a decrease in promoter function both in vitro and in vivo establishing it as a causative hereditary spherocytosis mutation. The dinucleotide deletion is located in the 5' untranslated region of the ANK-1 gene and disrupts the binding of TATA binding protein and TFIID, components of the preinitiation complex. We hypothesized that the nucleotides surrounding the mutation define an uncharacterized regulatory sequence. To test this hypothesis, we generated a library of more than 16,000 ANK-1 promoters with degenerate sequence around the mutation and cloned the functional promoter sequences after cell-free transcription. We identified the wild type and three additional sequences, from which we derived a consensus. The sequences were shown to be functional in cell-free transcription, transient-transfection, and transgenic mouse assays. One sequence increased ANK-1 promoter function 5-fold, while randomly chosen sequences decreased ANK-1 promoter function. Our results demonstrate a novel functional motif in the ANK-1 promoter.

Posttranslational control of transcription factor FixK 2 , a key regulator for the Bradyrhizobium japonicum –soybean symbiosis

Rhizobial FixK-like proteins play essential roles in activating genes for endosymbiotic life in legume root nodules, such as genes for micro-oxic respiration. In the facultative soybean symbiont, Bradyrhizobium japonicum, the FixK(2) protein is the key player in a complex regulatory network. The fixK(2) gene itself is activated by the 2-component regulatory system FixLJ in response to a moderate decrease of the oxygen tension, and the FixK(2) protein distributes and amplifies this response to the level of approximately 200 target genes. Unlike other members of the cAMP receptor protein family, to which FixK(2) belongs, the FixK(2) protein does not appear to be modulated by small effector molecules. Here, we show that a critical, single cysteine residue (C183) near the DNA-binding domain of FixK(2) confers sensitivity to oxidizing agents and reactive oxygen species. Oxidation-dependent inactivation occurs not only in vitro, as shown with cell-free transcription assays, but also in vivo, as shown by microarray-assisted transcriptome analysis of the FixK(2) regulon. The oxidation mechanism may involve a reversible dimerization by intermolecular disulfide-bridge formation and a direct, irreversible oxidation at the cysteine thiol, depending on the oxidizing agent. Mutational exchange of C183 to alanine renders FixK(2) resistant to oxidation, yet allows full activity, shown again both in vitro and in vivo. We hypothesize that posttranslational modification by reactive oxygen species is a means to counterbalance the cellular pool of active FixK(2), which would otherwise fill unrestrictedly through FixLJ-dependent synthesis.

Molecular and phenotypic traits of in-vitro-selected mutants of Bifidobacterium resistant to rifaximin

Nucleotide mutations inside a core region of the rpoB gene, encoding the β subunit of RNA polymerase, were found in rifaximin-resistant mutants of Bifidobacterium. Five different missense mutations of codons 513, 516, 522 and 529 were identified. Further aspects of rifaximin resistance were investigated, using Bifidobacterium infantis BI07 as a model strain. Partial resistance of RNA polymerase of a BI07 mutant at a rifaximin concentration >10 μg/mL was observed by cell-free transcription assay. Mass spectrometry detection of rifaximin in the cellular pellet of the BI07 resistant mutant, as well as changes in biosynthesis of saturated and cyclopropane fatty acids during growth, suggested a reduction in membrane permeability for the antibiotic moiety.

Molecular Mechanisms by Which Human Immunodeficiency Virus Type 1 Integrase Stimulates the Early Steps of Reverse Transcription

Reverse transcriptase (RT) and integrase (IN) are two essential enzymes that play a critical role in synthesis and integration of the retroviral cDNA, respectively. For human immunodeficiency virus type 1 (HIV-1), RT and IN physically interact and certain mutations and deletions of IN result in viruses defective in early steps of reverse transcription. However, the mechanism by which IN affects reverse transcription is not understood. We used a cell-free reverse transcription assay with different primers and compositions of deoxynucleoside triphosphates to differentially monitor the effect of IN on the initiation and elongation modes of reverse transcription. During the initiation mode, addition of IN stimulated RT-catalyzed reverse transcription by fourfold. The stimulation was specific to IN and could not be detected when the full-length IN was replaced with truncated IN derivatives. The IN-stimulated initiation was also restricted to the template-primer complex formed using tRNA(3)(Lys) or short RNA oligonucleotides as the primer and not those formed using DNA oligonucleotides as the primer. Addition of IN also produced a threefold stimulation during the elongation mode, which was not primer dependent. The stimulation of both initiation and elongation by IN was retained in the presence of an RT trap. Furthermore, IN had no effect on steps at or before template-primer annealing, including packaging of viral genomic RNA and tRNA(3)(Lys). Taken together, our results showed that IN acts at early steps of reverse transcription by increasing the processivity of RT and suppressing the formation of the pause products.

Novel Mechanism of β0 Thalassemia: Missense Mutation of the Last Nucleotide of β Globin Exon 1 (G->C) Leads to Undetectable Transcript and Mutant Peptide.

Hemoglobin Monroe (Hb Monroe) results from a point mutation (G->C) in the last nucleotide of β globin exon 1, which is also the penultimate nucleotide of codon 30 of the β globin mRNA (AGG->ACG/Arg->Thr). Hb Monroe was described seventeen years ago simultaneously by two groups: in an African American female with β thalassemia intermedia, wherein the thalassemia was thought to result from highly unstable peptide (Hemoglobin. 1989; 13:67); and in a North African female with compound β thalassemia (Proc Natl Acad Sci U S A. 1989; 86:1041) wherein the Hb Monroe mutation was thought to result in abnormal pre-mRNA splicing as detected in an in vitro cell-free transcription assay. We evaluated a 31-year old previously asymptomatic woman of Asian Indian (Bengali) descent, who presented with flu like symptoms and found to have low hemoglobin level (9.5 gm/dL), microcytosis (MCV 68 fL), moderately elevated liver enzymes and serum ferritin concentration of 3000 ng/ml. A liver biopsy revealed increased liver iron and significant fibrosis. Hemoglobin analysis, which was interpreted as compound heterozygosity for HbE/β0 thalassemia, revealed HbF: 51%, HbE: 43.2%, HbA2: 5.8%. β globin gene sequencing showed Hb Monroe and E mutations. The asymptomatic brother of the proband had borderline anemia (Hb 12 gm %), microcytosis (MCV 70 fl), HbF: 8.5 %, HbA: 87.2%, HbA2: 5.0% and was heterozygous for Hb Monroe mutation by Bme 15801 restriction enzyme analysis of genomic DNA. We set out to determine the molecular basis of the thalassemia phenotype associated with Hb Monroe mutation and whether this mutation in our subjects is present on African haplotype or had arisen independently. Since we could not detect the mutant peptide either in fresh hemolysate or reticulocyte enriched preparations; we expanded the peripheral blood erythroid progenitor cells in vitro of both proband and her brother. Hemoglobin analysis by both HPLC and mass spectrophotometry did not detect Hb Monroe peptide in the expanded cells. β globin cDNA from the reticulocytes and expanded erythroid progenitors was amplified using three different primer sets and no splice variants were detectable. Sequencing of the amplified cDNA revealed only normal β globin mRNA transcript. Other β globin gene mutations cis to Hb Monroe are being ruled out; to date, we have not found any promoter region or stop codon mutations, deletions or splicing mutations from promoter - 90 region to 3′ UTR including poly-A region. Haplotype analysis revealed a different haplotype from the two African American patients, indicating an independent origin of Hb Monroe mutation in our cases. Interestingly, both of our cases have elevated HbF, as did the two originally reported Hb Monroe patients (16.5 and 85%) and a currently unreported African American patient with sickle cell - β0 thalassemia due to Hb Monroe (11.4%). Hb F was also high in a group of nine patients reported with sickle cell - β0 thalassemia due to Hb Monroe (3.1%– 8.9%; Hemoglobin. 1998; 22:153). We conclude that this missense mutation, IVS1-1 (G->C/Arg 30 Thr) results in undetectable transcript and mutant Hb peptide leading to β0 thalassemia. The molecular mechanism of the undetectable mutant transcript is being investigated.

RNA polymerase III transcribes human microRNAs

Prior work demonstrates that mammalian microRNA (miRNA or miR) expression requires RNA polymerase II (Pol II). However, the transcriptional requirements of many miRNAs remain untested. Our genomic analysis of miRNAs in the human chromosome 19 miRNA cluster (C19MC) revealed that they are interspersed among Alu repeats. Because Alu transcription occurs through RNA Pol III recruitment, and we found that Alu elements upstream of C19MC miRNAs retain sequences important for Pol III activity, we tested the promoter requirements of C19MC miRNAs. Chromatin immunoprecipitation and cell-free transcription assays showed that Pol III, but not Pol II, is associated with miRNA genomic sequence and sufficient for transcription. Moreover, the mature miRNA sequences of approximately 50 additional human miRNAs lie within Alu and other known repetitive elements. These findings extend the current view of miRNA origins and the transcriptional machinery driving their expression.

The Role of the General Transcription Factor IIF in Androgen Receptor-Dependent Transcription

The androgen receptor (AR) is a member of the steroid receptor subfamily of nuclear receptors and is important for normal male sexual differentiation and fertility. The major transactivation function of the AR, termed activation function 1 (AF1), is modular in structure and has been mapped to the N terminus of the protein. To understand better the mechanisms whereby the AR activates transcription, we have established a novel cell-free transcription assay. This is based on the use of a dual reporter gene template, containing promoter proximal and distal G-less cassettes, which result in different size transcripts that can be easily detected and quantified. The promoter proximal transcript gives an indication of transcription initiation and promoter escape, whereas the relative levels of the distal transcript indicate elongation efficiency. The AR-AF1-Lex protein enhanced production of both transcripts whereas, in the absence of DNA binding, the AF1 domain squelched both initiation and elongation. Mutations in the transactivation domain that impaired transactivation and/or binding of the general transcription factor IIF (TFIIF) were found to reduce the ability of AR-AF1 to squelch transcription. Addition of recombinant TFIIF reversed squelching of the promoter-proximal but not the -distal G-less transcript, whereas addition of TATA-binding protein failed to reverse squelching of either transcript. Taken together, these results demonstrate that the AR N-terminal transactivation function, AF1, has the potential to regulate transcription at both the level of initiation and elongation, and that interactions with TFIIF are important during preinitiation complex assembly/open complex formation and/or promoter escape.

Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex.

Nuclear receptors modulate the transcription of genes in direct response to small lipophilic ligands. Binding to ligands induces conformational changes in the nuclear receptors that enable the receptors to interact with several types of cofactor that are critical for transcription activation (transactivation). We previously described a distinct set of ligand-dependent proteins called DRIPs, which interact with the vitamin D receptor (VDR); together, these proteins constitute a new cofactor complex. DRIPs bind to several nuclear receptors and mediate ligand-dependent enhancement of transcription by VDR and the thyroid-hormone receptor in cell-free transcription assays. Here we report the identities of thirteen DRIPs that constitute this complex, and show that the complex has a central function in hormone-dependent transactivation by VDR on chromatin templates. The DRIPs are almost indistinguishable from components of another new cofactor complex called ARC, which is recruited by other types of transcription activators to mediate transactivation on chromatin-assembled templates. Several DRIP/ARC subunits are also components of other potentially related cofactors, such as CRSP, NAT, SMCC and the mouse Mediator, indicating that unique classes of activators may share common sets or subsets of cofactors. The role of nuclear-receptor ligands may, in part, be to recruit such a cofactor complex to the receptor and, in doing so, to enhance transcription of target genes.

The role of phosphorylation in human estrogen receptor function

We have studied the role of phosphorylation of the human estrogen receptor (hER) at serine 118, which has been previously identified as a site important for transactivation. We have tested this transactivation in yeast and cell-free transcription assays, and have shown that mutation of serine 118 to alanine results in a 30–40% decrease in hER-dependent transcription. Furthermore, we investigated the functional significance of phosphorylation at this site by hormone binding and DNA binding. The mutation of serine 118 to alanine in the hER caused no decrease in its affinity for either estradiol or an ERE. The mutant receptor had an altered phosphorylation pattern when expressed in COS-1 and Sf9 cells, but not in HeLa cells. Our findings indicate that phosphorylation of serine 118 of the hER plays a role in regulating its transcriptional activity.

The HIV-1 Inducer of Short Transcripts Activates the Synthesis of 5,6-Dichloro-1-β-d-benzimidazole-resistant Short Transcripts in Vitro

The HIV-1 inducer of short transcripts (IST) is an unusual promoter element that activates the synthesis of short transcripts from the HIV-1 promoter as well as from heterologous promoters. While the DNA sequences constituting IST have been characterized in some detail, little is known about the biochemical mechanisms underlying IST activity. Here, we describe a cell-free transcription assay that faithfully reproduces the synthesis of IST-dependent HIV-1 short transcripts. As in vivo, formation of these short transcripts requires a functional IST element and is repressed in the presence of the viral trans-activator Tat. Short transcript and full-length transcript synthesis respond differently to variations in several reaction parameters, suggesting that the short and full-length transcripts are synthesized by transcription complexes with distinct biochemical properties. In particular, short transcript synthesis is resistant to the action of 5,6-dichloro-1-beta-D-benzimidazole, an inhibitor of transcript elongation. Formation of transcription complexes directed by the IST element may, therefore, not require the activity of a factor inhibited by 5, 6-dichloro-1-beta-D-benzimidazole, such as the TFIIH-associated or pTEFb kinases.

Molecular and kinetic basis for the mixed agonist/antagonist activity of estriol.

Estriol acts as a weak estrogen when administered in a single dose into immature or ovariectomized laboratory animals, but produces full estrogenic responses upon chronic administration. However, when estriol is injected together with estradiol it acts as an antiestrogen. We studied the dual agonist/antagonist properties of estriol, using recombinant human estrogen receptor (hER) in ligand-binding assay, cell-free transcription assay, electrophoretic mobility shift assay with cVitII estrogen response element (ERE), and ERE-Sepharose chromatography. We show that the weak estrogenic activity of estriol results from impaired hER-ERE interaction. The antiestrogenic activity of estriol was demonstrated in a cell-free transcription assay where it reduced estradiol-dependent transcription in a dose-dependent manner. Estriol interfered with estradiol-induced positive cooperative binding and receptor dimerization, and binding of hER complexes to ERE. These effects of estriol were maximal at a 10-fold molar excess over estradiol; under these conditions estradiol-dependent transcription was decreased by 85%, although [3H]estradiol binding was reduced by only 50%. We propose that when hER, estradiol, and estriol are coequilibrated, several receptor species are formed: unliganded hER monomers and dimers; estradiol-hER monomers and dimers, estriol-hER monomers and dimers; and presumably mixed estradiol-estriol dimers. Since estrogen-hER complexes bind cooperatively to ERE sequences, the concentrations of transcriptionally active complexes (estriol- and estradiol-hER dimers) are reduced to low levels that fail to bind cooperatively with ERE and initiate transcription. We discuss our results in relation to the massive estriol production during pregnancy and to the "Estriol Hypothesis" on the protective role for estriol in opposing carcinogenic effects of estradiol.

Molecular and Kinetic Basis for the Mixed Agonist/Antagonist Activity of Estriol

Estriol acts as a weak estrogen when administered in a single dose into immature or ovariectomized laboratory animals, but produces full estrogenic responses upon chronic administration. However, when estriol is injected together with estradiol it acts as an antiestrogen. We studied the dual agonist/antagonist properties of estriol, using recombinant human estrogen receptor (hER) in ligand-binding assay, cell-free transcription assay, electrophoretic mobility shift assay with cVitII estrogen response element (ERE), and ERE-Sepharose chromatography. We show that the weak estrogenic activity of estriol results from impaired hER-ERE interaction. The antiestrogenic activity of estriol was demonstrated in a cell-free transcription assay where it reduced estradiol-dependent transcription in a dose-dependent manner. Estriol interfered with estradiol-induced positive cooperative binding and receptor dimerization, and binding of hER complexes to ERE. These effects of estriol were maximal at a 10-fold molar excess over estradiol; under these conditions estradiol-dependent transcription was decreased by 85%, although[ 3H]estradiol binding was reduced by only 50%. We propose that when hER, estradiol, and estriol are coequilibrated, several receptor species are formed: unliganded hER monomers and dimers; estradiol-hER monomers and dimers, estriol-hER monomers and dimers; and presumably mixed estradiol-estriol dimers. Since estrogen-hER complexes bind cooperatively to ERE sequences, the concentrations of transcriptionally active complexes (estriol- and estradiol-hER dimers) are reduced to low levels that fail to bind cooperatively with ERE and initiate transcription. We discuss our results in relation to the massive estriol production during pregnancy and to the “Estriol Hypothesis” on the protective role for estriol in opposing carcinogenic effects of estradiol.

Competent transcription initiation by RNA polymerase II in cell-free extracts from xeroderma pigmentosum groups B and D in an optimized RNA transcription assay

The human autosomal recessive disease, xeroderma pigmentosum (XP), can result from mutations in any one of seven genes, designated XPA through XPG. Of these, the XPB and XPD genes encode proteins that are subunits of a general transcription factor, TFIIH, involved in both nucleotide excision repair (NER) and initiation of mRNA transcription by RNA polymerase II. In humans, mutation of the XPB or XPD gene impairs NER, resulting in hyper-sensitivity to sunlight and greatly increased skin tumor formation. However, no transcription deficiency has been demonstrated in either XP-B or XP-D. We have employed an optimized cell-free RNA transcription assay to analyze transcription activity of XP-B and XP-D. Although the growth rate was normal, the XP-B and XP-D cells contained reduced amounts of TFIIH. Extracts prepared from XP-B and XP-D lymphoblastoid cells exhibited similar transcription activity from the adenovirus major late promoter when compared to that in extracts from normal cells. Thus, we conclude that the XP-B and XP-D lymphoblastoid cells do not have impaired RNA transcription activity. We consider the possible consequences of the reduced cellular content of TFIIH for the clinical symptoms in XP-B or XP-D patients, and discuss a `conditional phenotype' that may involve an impairment of cellular function only under certain growth conditions.

Potentiation of human immunodeficiency virus type 1 Tat by human cellular proteins.

The Tat protein is a potent activator of human immunodeficiency virus type 1 transcription. Tat has been shown to act by increasing both transcription initiation and elongation, but a detailed understanding of its interaction with the transcriptional machinery is lacking. With the aim of isolating cellular proteins that interact with Tat and play a role in transactivation, we have reexamined its function in a cell-free transcription assay. Monitoring the appearance of transactivation after addition of purified Tat at intervals to the reaction mix revealed a lag of approximately 10 min before Tat is able to effect transactivation. Incubation of Tat in nuclear or cytoplasmic extracts of human cells was sufficient to eliminate the lag, but nuclear extract from a rodent cell line was inactive. The accelerating effect of the human cell extract could be abrogated by dilution, heat inactivation, or chromatographic depletion. We infer that Tat is potentiated for transactivation through interaction with a protein factor(s) that is specific to human cells.

P V.7 - P V.7 Nucleotide excision repair and initiation of RNA transcription in an optimized cell-free DNA repair and RNA transcription assay

P V.7 - P V.7 Nucleotide excision repair and initiation of RNA transcription in an optimized cell-free DNA repair and RNA transcription assay