Characterization of the trans-activation-responsive element of the parvovirus H-1 P38 promoter (original) (raw)
Related papers
Virology, 1992
In the parvovirus H-l P38 promoter, there are sequences identified as a TATA box, an SPl site, and a Vans-activation responsive element (tar). It was previously shown that the parvovirus H-l nonstructural protein NSl positively regulates the expression of the P38 promoter for the viral capsid protein gene via the tar. To characterize the tar element further, a series of single-point mutations of the tar was constructed and the mutants were compared to wild-type for the rrans-activation of the P38 promoter using a cat reporter gene. Most of the tar mutations had a negative effect on the P38 promoter and some of them reduced activity as much as 70%. However, when several mutants with multiplepoint mutations in the tar were tested, no significant additive effect was observed. We examined the function of the SPl site in the trans-activation of the P38 promoter by replacing the wild-type SPl sequence with synthetic DNA fragments, OSPl or 2SP1, containing no SPl or two SPl sites respectively, in a P38 construct with a cat reporter gene. The results indicate that P38 expression varies in proportion to the number of SPl sites, suggesting a role for the SPl site during trans-activation by NSl The role of the TATA box on the P38 promoter was also examined by mutagenizing TATA to CACG. The activity of this promoter was reduced to 43%. When a construct mutated at both the SPl and TATA box sites was tested for its activity, about 22% of the wild-type activity remained, implying that this remaining activity was contributed largely by the tar element. A model is proposed for how the tar element activates the wild-type and SPl-TATA minus promoters in the presence of NSl,
trans-Activation of parvovirus P38 promoter by the 76K noncapsid protein
Journal of Virology, 1985
The autonomously replicating parvoviruses contain a 5-kilobase linear single-stranded DNA genome that produces two noncapsid proteins, Ni and N2, and two overlapping capsid proteins, VP1 and VP2. To characterize the regulation of viral transcription, we began with a study of the promoter for the coat proteins (P38) at map unit 38. Various constructions containing the P38 promoter were fused to the bacterial gene for chloramphenicol acetyltransferase (cat), and the relative efficiency of expression was determined in the presence and absence of parvovirus gene products. Our results show that the P38 promoter is a weak promoter This work was supported by grant LB 506 85-56 from the state of Nebraska; Public Health Service grants RO1, CA26801, and CA36727, from the National Institutes of Health; and grant DBM-8444778 from the National Science Foundation. I thank L. Laimins for plasmids pAlocat and pSV2cat, Carol Wilson for technical assistance, and R. Hines, A. Rizzino, and E. Bresnick for reviewing the manuscript.
Journal of Virology
The NS-1 gene of the parvovirus minute virus of mice (MVM) (prototype strain, MVMp) was fused in phase with the sequence coding for the DNA-binding domain of the bacterial LexA repressor. The resulting chimeric protein, LexNS-1, was tested for its transcriptional activity by using various target promoters in which multiple LexA operator sequences had been introduced. Under these conditions, NS-1 was shown to stimulate gene expression driven by the modified long terminal repeat promoters (from the retroviruses mouse mammary tumor virus and Rous sarcoma virus) and P38 promoter (from MVMp), indicating that the NS-1 protein is a potent transcriptional activator. It is noteworthy that in the absence of LexA operator-mediated targeting, the genuine mouse mammary tumor virus and Rous sarcoma virus promoters were inhibited by NS-1. Together these data strongly suggest that NS-1 contains an activating region able to induce promoters with which this protein interacts but also to repress transcription from nonrecognized promoters by a squelching mechanism similar to that described for other activators. Deletion mutant analysis led to the identification of an NS-1 domain that exhibited an activating potential comparable to that of the whole polypeptide when fused to the DNA-binding region of LexA. This domain is localized in the carboxy-terminal part of NS-1 and corresponds to one of the two regions previously found to be responsible for toxicity. These results argue for the involvement of the regulatory functions of NS-1 in the cytopathic effect of this parvovirus product.
1992
To further define the transcriptional regulation of the P38 promoter in the minute virus of mice (MVM) genome, we constructed a series of internal deletion and linker scanning mutations. The mutant P38 constructs were assayed for transcriptional activity in vitro by primer extension analysis with nuclear extracts from murine A92L fibroblasts. Mutations which disrupted the GC box and TATA box severely reduced transcription in vitro. DNase I footprinting analysis confirmed that the murine transcription factor Spl bound to the GC box; however, no factors were observed interacting with a putative transcriptional activation regulatory element, termed the TAR element. The linker scanning mutations were analyzed in vivo by using a chloramphenicol acetyltransferase expression assay system, in both the presence and absence of constructs expressing the viral nonstructural protein, NS1. The ability of NS1 to transactivate the P38 promoter (up to 1,000-fold) depended entirely on the presence of...
Journal of General Virology, 1988
The genome of the autonomous parvovirus minute virus of mice (MVM) is organized in two overlapping transcription units: the genes coding for the two non-structural proteins (NS-1 and NS-2) are transcribed from a promoter (P04) located at map unit 4, whereas the promoter controlling the capsid protein genes (P39) lies at map unit 39. We studied the effect of viral proteins on the activity of the P39 promoter in vivo. By sitedirected mutagenesis we constructed clones encoding only one of the two NS proteins. The activity of the P39 promoter was measured in HeLa or EL-4 cells transfected with these clones, either by an RNase protection assay or by following the expression of a reporter gene, CAT (which codes for chloramphenicol acetyltransferase), placed under the control of this promoter. We found that the P39 promoter of strain MVMi is activated in trans by a viral gene product, and evidence to suggest that NS-1 is the only viral gene product responsible for this trans-activation. We also determined that the mechanism of trans-activation is very rapid, since all species of viral mRNAs appear together in non-synchronized infected EL-4 cells within a 2 h interval.
Journal of Biotechnology, 2013
The structure of eukaryotic promoter is modular, consisting of different sub-domains . Cross-talk among these sub-domains and effective combinatorial interactions between the specific cis-element/s with respective transfactor/s usually control the strength and tissue specificity of the promoter. Further, specificity/inducibility of promoter can be modified by altering its genetic architecture through 'cis-rearrangement and 'swapping of sub-domains' . The fundamental motivation behind developing such modified promoters lies in the belief that the swapping/shuffling of the upstream activation sequences carrying a specific set of cis-elements that bind to a particular trans-factor from one promoter to the other containing a TATA sequence that might result in a novel chimeric regulatory module, the modified promoter. Such engineered/ modified promoters could be of great importance over natural (parent) promoters for not only do they hold better potential for ensuring efficient gene expression in plant under both biotic and abiotic stresses, but they also provide evidence for better understanding of the promoter structure and function .
Virology, 1992
The parovivirus H-l P38 promoter contains a trans-activation responsive element (tar). It was previously shown that the parvovirus H-l nonstructural protein NSl positively regulates the expression of the P38 promoter for the viral capsid protein gene via the tar (Rhode and Richard, 1987,J. Viral. 61, 2807-2815). To characterize the mechanism of trans-activation by the tar, we used gel shift assays to demonstrate that there exist proteins in virus-infected cellular extracts which have higher binding activity than that found in mock-infected extracts. These observations in vitro are consistent with the expression by P38 constructs with the wild-type promoter linked to a reporter gene, chloramphenicol acetyl transferase (cat), in viva. We also provide evidence that the protein(s)-tar complex has a molecular mass of approximately 75 kDa in an SDS-polyacrylamide gel, which is less than NSl, and this complex cannot be precipitated by NSl antibody, which suggests that NSl mediates the trans-activation by inducing an alteration in the binding activity of some cellular protein(s) in an indirect manner. These data support our previous hypothesis for the activation of the P38 promoter, in which the trans-activator(s) interacts with the tar effectively in the presence of NSl, leading to the formation of the transcription initiation complex by protein-protein associations (Gu, Chen, and Rhode, 1992, Virology 187, 1 O-l 7).
Proc. Natd Acad. Sci. USA Vol. 78, No. 8, pp. 4936-4940, 1981
Downstream placement of a strong transcrip-tional termination signal has made possible the cloning of bacte-riophage T5 promoters known to exhibit high signal strength. The cloning system constructed contains two easily assayable indicator functions whose expression is controlled by the integration of promoters and terminators, respectively. By assessing transcription within the indicator regions, the efficiency of promoters as well as termination signals can be determined in vitro and in vivo. The efficiency of interaction between Escherichia coli RNA polymerase and transcriptional promoters of E. coli varies within a wide range when measured in vitro (1). For unregu-lated promoters, the rate ofcomplex formation in vitro reflects promoter strength in vivo (1, 2). However, despite the identification of more than 80 different promoter sequences and extensive study of promoter-RNA polymerase interactions (for survey, see refs. 3-5), the contribution of specific structural features to the functional activity of such sequences is not understood. Promoters from various bacterial and viral sources have been cloned in E. coli, and their signal strength in vivo has been studied by using expression from distal promoterless sequences encoding (3-galactosidase ((-Gal) or other proteins (6, 7) as an indicator of promoter activity. Attempts to clone small DNA fragments carrying the strong promoters of bacteriophage T5, which in vitro far exceed other promoters in the rate ofcomplex formation with RNA polymerase and the rate of initiation of RNA synthesis (1, 2), have been unsuccessful; however, fragments of T5 DNA containing both a strong promoter and a strong termination signal have been cloned (8). Subsequently, electron microscope analysis has shown that transcriptional regions of several E. coli plasmids are organized in well-defined units where termination signals appear to balance transcription initiated at promoters ofdifferent strengths (9). Together, these findings suggested that the cloning of strong promoter signals from phage T5 or other sources might require the downstream placement of comparably strong termination signals. We report here the construction and analysis of bacterial plasmid vectors that enable the cloning of promoters of high signal strength; such cloning is made possible by the positioning ofa transcriptional termination signal downstream from the site ofinsertion ofsuch promoters. The constructed plasmids, which allow estimation ofthe strength ofpromoter signals in vitro and in vivo, contain indicator genes in positions that also permit selection for termination signals. Using these vectors, we have isolated a library of T5 promoter sequences suitable for biochemical and physical investigations of promoter function and also potentially useful for achieving high-level transcription of heterologous genes introduced distal to the promoter signals. MATERIALS AND METHODS Restriction endonucleases, E. coli DNA polymerase, and bac-teriophage T4 DNA ligase were purchased from several commercial sources, and reactions were carried out as suggested by the supplier. EcoRI synthetic linker and adapter sequences were obtained from Collaborative Research (Waltham, MA). Phagefd DNA (replicative form) and plasmid pAD16/30 containing a 28-base-pair (bp) HindIII/BamHI adapter sequence were gifts from H. Schaller. lac repressor was a gift from A. Riggs. Isolation of bacteriophage T5 DNA (2), plasmid DNA (10), E. coli RNA polymerase (2), and termination factor rho (11) have been described previously. The binding of RNA polymerase to promoters and subsequent analysis of the complexes by nitrocellulose filter binding have been described (2). Identification and isolation of lac operator containing DNA fragments by repressor binding utilized the procedure of Riggs et al (12). The conversion of protruding 5' single-stranded DNA extensions to blunt ends and DNA lig-ation reactions have been described (13). Synthetic linker and adapter sequences were present in 3-to 10-fold excess relative to the various DNA fragments. Transformation ofE. coli strains C600r-m' (our laboratory collection), the M15 deletion-mutant DZ 291 (obtained from A. V. Fowler), and BMH71-18, an MiS derivative carrying the laCjq mutation (obtained from B. Muel-ler-Hill), was carried out as described (14). Selection oftransformants involved plating on LB plates containing chloramphenicol (Cm, 20 Ag/ml), ampicillin (Ap, 100 gg/ml), or varying amounts of tetracycline (Tc; 2-70 tug/ml). Selection for presence of the lac operator or production of the a fragment of (3-Gal was carried out on plates containing the antibiotic plus 5-bromo-4-chloro-3-indolyl (3-O-galactoside at 40 ug/ml (15). Induction of lac expression by isopropylthio-galactoside was as described (15). In vitro and in vivo RNA was prepared and analyzed as described previously (1) except that [a-32P]UTP and [32P]phosphate were used for labeling in vitro and in vivo, respectively. In vivo RNA was isolated from plas-mid-containing C600 cells after a 10-min labeling period. RESULTS Experimental Strategy. We have constructed a family of plasmids (Fig. 1) that carry two DNA segments that can be brought under the control of a single promoter and are separated by an endonuclease cleavage site suitable for the cloning Abbreviations: bp, base pair(s); Cm, chloramphenicol; Tc, tetracycline; Ap, ampicillin; (B-Gal, 3-galactosidase. 4936 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Molecular and Cellular Biology, 2005
Downstream elements are a newly appreciated class of core promoter elements of RNA polymerase IItranscribed genes. The downstream core element (DCE) was discovered in the human -globin promoter, and its sequence composition is distinct from that of the downstream promoter element (DPE). We show here that the DCE is a bona fide core promoter element present in a large number of promoters and with high incidence in promoters containing a TATA motif. Database analysis indicates that the DCE is found in diverse promoters, supporting its functional relevance in a variety of promoter contexts. The DCE consists of three subelements, and DCE function is recapitulated in a TFIID-dependent manner. Subelement 3 can function independently of the other two and shows a TFIID requirement as well. UV photo-cross-linking results demonstrate that TAF1/TAF II 250 interacts with the DCE subelement DNA in a sequence-dependent manner. These data show that downstream elements consist of at least two types, those of the DPE class and those of the DCE class; they function via different DNA sequences and interact with different transcription activation factors. Finally, these data argue that TFIID is, in fact, a core promoter recognition complex. . † Supplemental material for this article may be found at http://mcb .asm.org/. 9674 pH 7.9, 10 mM HEPES-KOH, pH 8.0, 4 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 100 g/ml bovine serum albumin, 10 mM dithiothreitol, and 250 M recombinant nucleoside triphosphates. Ad E3 in vitro transcription mixtures also contained baculovirus-expressed highly purified Sp1 and a partially purified mediator fraction in order to increase transcription signals .
Journal of virology, 1993
The simian virus 40 large T antigen is a promiscuous transcriptional activator of many viral and cellular promoters. We show that the promoter structure necessary for T antigen-mediated transcriptional activation is very simple. A TATA or initiator element is required, in addition to an upstream factor-binding site, which can be quite variable. We found that promoters containing an SP1-, ATF-, AP1-, or TEF-I-binding site, in conjunction with a TATA element, can all be activated in the presence of T antigen. In addition, preference for specific TATA elements was indicated. Promoters containing the HSP70 TATA element functioned better than those with the adenovirus E2 TATA element, while promoters containing the simian virus 40 (SV40) early TATA element failed to be activated. In addition, simple promoters containing the initiator element from the terminal deoxynucleotidyltransferase gene could be activated by T antigen. The SV40 late promoter, a primary target for T antigen transcrip...