Showing papers on "Transcription factor published in 1984"

Cyclic AMP receptor protein: role in transcription activation

Abstract: The structure of this pleiotropic activator of gene transcription in bacteria and its interaction sites at promoter DNA's as well as the role of this protein in the RNA polymerase-promoter interactions are reviewed.

Multiple specific contacts between a mammalian transcription factor and its cognate promoters

Abstract: The human transcription factor Sp1 binds upstream of certain viral and cellular promoters and activates initiation of RNA synthesis from these promoters by RNA polymerase II. The Sp1 binding sites of both simian virus 40 and a related monkey promoter contain multiple copies of the sequence GGGCGG, three or four of these sequences forming close contacts with Spl. The clustered contacts fall on one strand of the DNA and are arranged similarly in the major groove of the DNA helix in both promoters.

The Role of Stable Complexes that Repress and Activate Eukaryotic Genes

Abstract: The state of activity or repression of 5S RNA genes is built into the chromatin in the form of stable complexes. The characteristics and composition of these complexes will be described. Stable transcription complexes that activate 5S RNA genes consist of multiple factors bound tightly to a control region of the gene. Repressed 5S RNA genes do not have these factors bound to them; their repressed state is maintained by nucleosomes.

Isolation of a class C transcription factor which forms a stable complex with tRNA genes.

Abstract: A yeast extract was fractionated to resolve the factors involved in the transcription of yeast tRNA genes. An in vitro transcription system was reconstituted with two separate protein fractions and purified RNA polymerase C (III). Optimal conditions for tRNA synthesis have been determined. One essential component, termed tau factor, was partially purified by conventional chromatographic methods on heparin-agarose and DEAE-Sephadex; it sedimented as a large macromolecule in glycerol gradients (mol. wt. approximately 300 000). tau factor was found to form a stable complex with the tRNA gene in the absence of other transcriptional components. Complex formation is very fast, is not temperature dependent between 10 degrees C and 25 degrees C and does not require divalent cations. The factor-DNA complex is stable for at least 30 min at high salt concentration (0.1 M ammonium sulfate). These results indicate that gene recognition by a specific factor is a primary event in tRNA synthesis.

Binding of Xenopus transcription factor A to 5S RNA and to single stranded DNA

Abstract: Footprint competition assays are utilized to study the binding of Xenopus transcription factor A to a variety of single-stranded nucleic acids. The addition of Xenopus oocyte, yeast, or wheat germ 5S RNA as footprint competitors reveals that factor A binds these 5S RNAs with similar affinity. In contrast, factor A does not bind to E.coli 5S RNA or wheat germ tRNA in this assay. Factor A binding to single stranded DNA is also examined using footprint competition. Factor A binds preferentially to non-specific single stranded (M13) DNA versus double stranded (pBR322) DNA. Factor A binds equally well to single stranded DNA fragments containing either the coding or non-coding strands of the 5S RNA gene. Using single stranded M13 DNA as a competitor, the factor A-5S RNA gene complex is found to dissociate with a half-life of 5-6 min.

An enhancer sequence from bovine papilloma virus DNA consists of two essential regions

Abstract: A comprehensive structural analysis of an enhancer sequence from bovine papilloma virus DNA is presented based on the construction and functional analysis of 20 mutant derivatives. The results, obtained in CV-1 tissue culture cells, show that this enhancer is a small genetic element--only 40 bp in length--that contains two essential regions. The two regions exhibit homology to each other and to DNA fragments from other viral genomes that also act as enhancers in the assay used. However, there is a certain latitude in the sequences that have enhancer activity in CV-1 cells, even within the critical regions. The results are discussed with respect to the model that enhancers are binding sites for tissue specific transcription factors. The formation of Z-DNA might be involved in the enhancement process. However, single base pair transitions in an 8 base pair stretch of alternating purines and pyrimidines within the BPV enhancer which conserve this pattern destroy enhancer function.

Regulation of polyomavirus transcription by large tumor antigen.

Abstract: We have analyzed the regulation of viral transcription by the large tumor antigen in cells infected by several viable deletion and insertion mutants of polyomavirus. We find that deletion of the early promoter "TATA box" and associated large tumor antigen binding site has only a small effect on the balance of early and late mRNAs. Furthermore, transcription of a polyomavirus containing a heterologous adenovirus promoter in place of the normal TATA box and cap sites is regulated by the large tumor antigen. We conclude that repression of polyomavirus early transcription cannot occur simply by binding of the large tumor antigen to DNA sequences at the site of transcription initiation and must involve the interaction of the large tumor antigen binding at other sites.

Glucocorticoid inhibition of initiation of transcription of the DNA encoding rRNA (rDNA) in lymphosarcoma P1798 cells.

Abstract: Cell-free extracts of lymphosarcoma P1798 cell culture lines support faithful initiation upon the cloned mouse DNA encoding rRNA (rDNA) promoter, whereas extracts from cells treated for 16 hr with 0.1 microM dexamethasone cannot. Extracts from both sources transcribe the cloned 5S RNA gene in vitro and mixing experiments further demonstrate that inhibition of transcription of rDNA in vitro is not due to nucleases or inhibitors of transcription present in extracts from glucocorticoid-treated cells. Incubation of extracts from control cells at 45 degrees C for 15 min inactivates RNA polymerase I and abolishes transcription. Activity can be restored by the addition of partially purified RNA polymerase I from control cells and hormone-treated cells. Moreover, extracts from hormone treated cells can be reconstituted by the addition of a partially purified, heat-stable transcription factor from control cells.

Simian virus 40 guanine-cytosine-rich sequences function as independent transcriptional control elements in vitro.

Abstract: We have recently shown that DNA sequences located within the simian virus 40 (SV40) G-C-rich, 21-base-pair repeats constitute an important transcriptional control element of the SV40 late promoter (Brady et al., Mol. Cell. Biol. 4:133-141, 1984). To gain further insight into the mechanism by which the SV40 G-C-rich repeats function, we have analyzed the transcriptional properties of several recombinant DNAs. The results presented in this report suggest that the SV40 G-C-rich sequences can function as independent RNA polymerase II transcriptional-control elements. In vitro competition studies demonstrated that sequences within the G-C-rich, 21-base-pair repeats, in the absence of either the SV40 early or late -25 transcriptional-control signals or the major RNA initiation sites, efficiently competed for transcription factors required for SV40 early and late RNA synthesis. Our transcription studies also demonstrated that in the absence of contiguous SV40 transcription control sequences, G-C-rich sequences stimulated initiation of transcription in a bidirectional manner, from proximally located sequences. Finally, we demonstrated that the 21-base-pair-repeat region can stimulate in vitro transcription from the heterologous adenovirus 2 major late promoter.

Mouse mammary tumor virus: transcriptional control and involvement in tumorigenesis.

Abstract: Publisher Summary This chapter describes the concept of transcriptional control in tumorigenesis. Hormonal regulation of gene expression has been studied in a variety of systems. Advances in the understanding of the mechanism of hormonal regulation are dependent upon the availability of hormone-responsive cell culture systems and molecular clones of hormonally regulated genes. As a functional glucocorticoid receptor is required for induction in transfected cells and because this induction is rapid and protein synthesis independent, it is reasonable to assume that there is a direct interaction of the receptor with the regulated gene. This interaction results in an increase in the rate of transcription. The glucocorticoid receptor assumes the role of a transcription factor and seems to focus correct initiation of RNA in the MMTV-LTR. Several possible molecular mechanisms, involving proviral insertional mutagenesis and subsequent mammary tumorigenesis, can be envisaged. The experiments leading to a further molecular understanding will be aimed at the identification of the genes activated by MMTV integration and a characterization of their function.

5S RNA structure and interaction with transcription factor A. 2. Ribonuclease probe of the 7S particle from Xenopus laevis immature oocytes and RNA exchange properties of the 7S particle

Abstract: The 5S RNA complexed in the 7S particle of immature Xenopus laevis oocytes was 32P labeled at its 3' end and then subjected in situ to partial digestion using ribonucleases T1, T2, A, and V1 in order to study the conformation of the complexed RNA and its interaction with the transcription factor A (TFIIIA). Digested samples were gel purified to retrieve 5S RNA that was still complexed with the transcription factor protein, and the cleavages in these RNAs were analyzed on sequencing gels. The RNA associated with the 7S particle is very susceptible to ribonuclease activity despite the presence of the protein. Also, the 5S RNA in the 7S particle is in a different conformation from renatured Xenopus laevis (Xlo) 5S RNA and appears to have less secondary structure predominantly in the stem that includes helices IV and V. A species of native Xlo 5S RNA which was isolated from 7S particle preparations under nondenaturing conditions revealed a conformation that was more similar to the 5S RNA in the 7S particle than to renatured 5S RNA. Comparison of data from partial ribonuclease digestions performed on renatured 5S RNA, on the native 5S RNA, and on the complexed 5S RNA allowed us to approximate sites of protein-induced structural change in the complexed 5S RNA, which may signal protein interaction domains. These sites include the 5' side of helices III and V. In another approach to the study of 5S RNA-TFIIIA interactions, we have observed that incubation of 32P-labeled Xlo 5S RNA with 7S particles results in the incorporation of labeled RNA into 7S particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of an RNA polymerase III transcription factor with a ribonucleoprotein complex recognized by autoimmune sera

Abstract: RNA polymerase III transcription can be inhibited in vitro by two sera from patients with autoimmune diseases. The first serum, designated anti-SS-B (or La), has antibodies directed against a 50,000 dalton polypeptide that is part of a larger ribonucleoprotein complex. The second serum, designated anti-SpNo, recognizes a target antigen polypeptide of greater than 100,000 daltons as well as the SS-B antigen. Both sera selectively remove required transcription factors from the transcription extract, and inhibition can be rescued by the addition of a HeLa S100 extract to the depleted transcription system. The HeLa S100 extract was sequentially fractionated by ion-exchange chromatography on DEAE-cellulose and phosphocellulose. The high salt eluate from the latter column was also able to rescue the anti-SS-B inhibition as was the immunoaffinity-purified SS-B ribonucleoprotein complex isolated from HeLa, Xenopus or rabbit thymus. Immunoblots of the active fractions indicated that all contained the SS-B immunoreactive polypeptide, but probes of replica filters for DNA-binding suggested that the transcription factor is not the SS-B antigen but a 64,000 dalton polypeptide component of the antigen ribonucleoprotein complex. SS-B is itself an RNA-binding protein and could be shown to bind nascent 5S RNA transcripts in vitro. Differential ammonium sulfate precipitation and DNA cellulose chromatography has confirmed that a group of 64-68 K dalton polypeptides are components of the SS-B ribonucleoprotein complex associated with transcription factor activity.

The absence of a human-specific ribosomal DNA transcription factor leads to nucleolar dominance in mouse greater than human hybrid cells.

Abstract: The basis for nucleolar dominance in mouse-human cell hybrids which contained all of the mouse chromosomes but an incomplete set of human chromosomes (M greater than H) was examined at the molecular level. S1 mapping data showed that these cells had the expected levels of steady-state rRNA transcribed from mouse ribosomal gene (rDNA) transcription units but undetectable levels of rRNA derived from the human rDNA transcription templates that are also present. RNA polymerase I-dependent, cell-free transcription extracts were made from three hybrid lines and were found to be capable of transcribing cloned rDNA templates of mouse but not human origin. Partially purified human factors required for rDNA transcription in vitro were added to the M greater than H extracts. One fraction with almost no RNA polymerase I activity conferred on these hybrid cell extracts the ability to transcribe a human rDNA template. These rescue experiments suggested that this required human-specific rDNA transcription factor(s) was effectively absent from the lines we examined and could account for nucleolar dominance in M greater than H hybrid cells.

Cytoplasmic and secreted Saccharomyces cerevisiae invertase mRNAs encoded by one gene can be differentially or coordinately regulated.

Abstract: A single structural gene, SUC2, encodes both secreted and cytoplasmic invertase in Saccharomyces cerevisiae. It is known that the unprocessed polypeptides which differ by a secretion signal sequence are encoded by separate mRNAs. This unusual transcriptional organization raises the question as to the degree to which the transcripts can be independently regulated. To define a system for studying this problem, we examined invertase transcription after various physiological perturbations of cells: rapid catabolite derepression, heat shock, and cell cycle arrest. With each treatment, fluctuations in mRNA levels for both cytoplasmic and secreted invertase were observed. We concluded that (i) catabolite-derepressed synthesis of the mRNAs occurs rapidly after a drop in glucose, is a sustained response, and does not require de novo protein synthesis; (ii) heat shock transcription of both invertase mRNAs is, in contrast, a brief and transient response requiring de novo protein synthesis; and (iii) alpha-mating hormone treatment (G1 phase arrest and release) results in regular and coordinated synthesis of both mRNAs midway between rounds of histone mRNA synthesis. We propose that invertase mRNA regulation involves constitutively synthesized transcriptional factors (observed during catabolite derepression) and transient factors (observed during heat shock and possibly during synchronous growth). Moreover, the mRNA levels for secreted and cytoplasmic invertase can be independently regulated.

Initiation and termination signals for transcription in bacteriophage M13

Abstract: Transcription of the infrequently expressed phage M13 genome domain, comprising genes III, VI, I and IV, has been studied in detail by hybridization and S1-nuclease mapping studies. The contiguous genes III and VI are transcribed via an 1800 nucleotide-long RNA molecule that is initiated at a promoter which overlaps with the Rho-independent termination signal between genes III and VIII. Its synthesis is terminated at a Rho-dependent terminator in the proximal part of gene I. Transcription of gene I is not mediated by an independent promoter but most probably by read-through of RNA-polymerase through this terminator. Transcription of gene IV is accomplished by synthesis of four distinct RNAs of about 1500 to 1680 nucleotides long which are initiated at a promoter located immediately in front of gene IV. Termination of these transcripts is generated at least four different sites located in tandem within the intergenic region between genes IV and II.

Structural requirements for the interaction of 5S rRNA with the eukaryotic transcription factor IIIA

Abstract: In order to study the binding of the eukaryotic transcription factor IIIA to heterologous 5S rRNAs with a low degree of overall sequence conservation (less than 20%) we have utilized a transcription competition assay involving eubacterial, archaebacterial and eukaryotic 5S rRNAs. All the molecules inhibit Xenopus 5S rRNA transcription specifically, which suggests that only a small amount of specific conserved RNA sequences, if indeed any, are essential for the interaction of the transcription factor with the 5S rRNA molecule, whereas universal 5S rRNA secondary structure elements seem to be required. A fragment of Xenopus laevis oocyte 5S rRNA (nucleotides 41-120), which partially maintains the original 5S rRNA structure, also competes for TF III A. In vitro transcription of a naturally occurring mutant of the Xenopus laevis oocyte 5S rRNA gene, the pseudogene, which carries several point mutations within the TF III A binding domain is equally inhibited by exogenous Xenopus 5S rRNA.

Homologous globin cell-free transcription system with comparison of heterologous factors.

Abstract: Mouse erythroleukemia (MEL) cells provide a useful model system to examine the regulation of globin gene expression. MEL cells ordinarily do not express globin genes, but in the presence of inducers, such as dimethyl sulfoxide or hexamethylene bisacetamide, they mimic erythroid differentiation. We have developed a cell-free transcription system from uninduced MEL cells to determine the requirements for mRNA synthesis. The MEL system directs accurate transcription of adenovirus type 2 major late DNA and mouse betamaj-globin with an efficiency comparable to those of HeLa and KB cell extracts. Using the procedure of Matsui et al. (T. Matsui, J. Segall, P.A. Weil, and R.G. Roeder, J. Biol. Chem. 255:11992-11996, 1980), we have isolated three active fractions from both MEL and HeLa cell extracts which are required for accurate transcription and have shown that equivalent fractions from MEL and HeLa cell extracts are interchangeable. Our findings suggest that the components required for initiation of transcription are similar in different cell types, at least to the extent that they can be assayed in these in vitro systems.

Transcription factor binding is limited by the 5'-flanking regions of a Drosophila tRNAHis gene and a tRNAHis pseudogene

Abstract: We determined the sequence of a Drosophila tRNA gene cluster containing a tRNAHis gene and a tRNAHis pseudogene in close proximity on the same DNA strand. The pseudogene contains eight consecutive base pairs different from the region of the bona fide gene which codes for the 3' portion of the anticodon stem of tRNAHis. The tRNAHis gene is transcribed efficiently in Drosophila Kc cell extract, whereas the pseudogene is not. The pseudogene is also a much poorer competitor than the real gene in a stable transcription complex formation assay, even though the sequence alteration in the pseudogene does not affect the sequence or spacing of the putative internal transcription control regions. Recombinant clones were constructed in which the 5'-flanking regions are exchanged. The transcription efficiencies and competitive abilities of the recombinant clones resemble those of the genes from which the 5' flank was derived; for example, the tRNAHis pseudogene with the 5'-flanking sequence of the tRNAHis gene is now efficiently transcribed. Deletion analysis of the pseudogene 5' flank failed to uncover an inhibitory element. Deletion analysis of the real gene showed very high dependence on the presence of the wild-type 5'-flanking sequence for factor binding to the internal control regions and stable complex formation. The 5'-flanking sequence of a Drosophila tRNAArg gene active in the Drosophila Kc cell extract does not restore transcriptional activity or stable complex formation. The tRNAHis gene and pseudogene behave atypically in HeLa cell extract. Both genes compete for HeLa transcription factors, but neither of them is efficiently transcribed. Removal of the 5'-flanking sequences of each gene and replacement with various sequences, including the tRNAArg gene 5' flank, does not allow increased transcription in HeLa cell extract.

Kinetic analysis of mutations affecting the cII activation site at the PRE promoter of bacteriophage lambda

Abstract: Abortive initiation and run-off transcription assays were used to study the effects of cy mutations on activation of the phage lambda PRE promoter by cII gene product. Six point mutations in the repeated T-T-G-C sequences that flank the -35 consensus region of PRE decreased the apparent affinity of the promoter for cII protein by factors of 4-16 relative to the wild-type affinity. Kinetic analyses of transcription initiation in the presence and absence of cII protein demonstrated that five of the six mutations did not significantly affect the intrinsic interaction of RNA polymerase with PRE. Thus, these mutations differ from other cy mutations, including those in the -35 consensus region, which affect the formation of polymerase-PRE closed complexes or the isomerization of closed complexes to open complexes but do not affect the binding of cII protein. A sixth T-T-G-C mutation, cy3001, may affect intrinsic initiation by RNA polymerase as well as cII binding.

Transcription analysis of the lexA gene of Escherichia coli: attenuation and cotranscription with the neighboring region.

Abstract: The lexA gene of Escherichia coli encodes a repressor of the genes whose expression is induced by the agents that result in DNA damage. In vivo transcripts of the lexA gene consisted of two species; mRNA-1 of 673 bases and mRNA-2 of approximately 3,000 bases. The transcription in vivo started at a site which was two-base pairs downstream from the in vitro initiation site reported previously. The majority of the transcription stopped at a series of T residues preceeded by a dyad symmetry located immediately after the lexA gene. A small fraction of the transcription passed through the termination site to form the mRNA of downstream gene(s) which would be related to the "SOS functions".