Showing papers on "Transcription factor published in 1985"

Repetitive zinc‐binding domains in the protein transcription factor IIIA from Xenopus oocytes.

Abstract: The 7S particle of Xenopus laevis oocytes contains 5S RNA and a 40-K protein which is required for 5S RNA transcription in vitro. Proteolytic digestion of the protein in the particle yields periodic intermediates spaced at 3-K intervals and a limit digest containing 3-K fragments. The native particle is shown to contain 7-11 zinc atoms. These data suggest that the protein contains repetitive zinc-binding domains. Analysis of the amino acid sequence reveals nine tandem similar units, each consisting of approximately 30 residues and containing two invariant pairs of cysteines and histidines, the most common ligands for zinc. The linear arrangement of these repeated, independently folding domains, each centred on a zinc ion, comprises the major part of the protein. Such a structure explains how this small protein can bind to the long internal control region of the 5S RNA gene, and stay bound during the passage of an RNA polymerase molecule.

Bidirectional SV40 transcription mediated by tandem Sp1 binding interactions.

Abstract: The 21-base pair repeat elements of the SV40 promoter contain six tandem copies of the GGGCGG hexanucleotide (GC-box), each of which can bind, with varying affinity, to the cellular transcription factor, Sp1. In vitro SV40 early RNA synthesis is mediated by interaction of Sp1 with GC-boxes I, II, and III, whereas transcription in the late direction is mediated by binding to GC-boxes III, V, and VI.

Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor.

Abstract: In enteric bacteria the products of two nitrogen regulatory genes, ntrA and ntrC, activate transcription of glnA, the structural gene encoding glutamine synthetase, both in vivo and in vitro. The ntrC product (gpntrC) is a DNA-binding protein, which binds to five sites in the glnA promoter-regulatory region and appears to activate transcription initiation. Using as an assay the stimulation of glnA transcription in a coupled in vitro transcription-translation system, we have partially purified the ntrA gene product (gpntrA). The following evidence is consistent with the view that gpntrA is a sigma subunit for RNA polymerase: (i) The gpntrA activity copurifies with the sigma 70 holoenzyme (E sigma 70) and core (E) forms of RNA polymerase through several steps but can be separated from them by chromatography on heparin agarose. (ii) After further purification by molecular sieve chromatography, the partially purified gpntrA fraction allows transcription of glnA from the same startpoint used in vivo; transcription is dependent on gpntrC and on added E. The gpntrA fraction does not allow transcription from promoters that we have used as controls, including lacUV5. E sigma 70 has the reverse specificity.

Transcription of glnA by purified Escherichia coli components: core RNA polymerase and the products of glnF, glnG, and glnL

Abstract: We have shown that the purified glnF (ntrA) product of Escherichia coli binds to core RNA polymerase. Together these proteins initiated transcription at the nitrogen-regulated promoter glnAp2 on a supercoiled template. The initiation of transcription at glnAp2 on a linear template required in addition NRI, the product of glnG (ntrC), and NRII2302, the product of a mutant allele of glnL (ntrB). These results identify the glnF product as a new sigma factor specifically required for the transcription of nitrogen-regulated and of nitrogen-fixation promoters. We propose rpoN as the proper designation for glnF, and sigma 60 for its product. Our results indicate that sigma 60 RNA polymerase recognizes the nitrogen-regulated/nitrogen-fixation promoter consensus sequence C-T-G-G-Y-A-Y-R-N4-T-T-G-C-A. Initiation of transcription in the intact cell appears to require in addition the active form of NRI, the product of glnG. Conversion of NRI to its active form is apparently brought about by NRII, the product of glnL, in response to nitrogen deprivation.

Sp1 binds to promoter sequences and activates herpes simplex virus ‘immediate-early’ gene transcription in vitro

Abstract: During a herpes simplex virus (HSV-1) lytic infection, three classes of viral genes are transcribed in a temporally regulated manner1–5. The HSV-1 ‘immediate-early’ (IE) promoter sequences contain multiple copies of a hexanucleotide sequence, GGGCGG, known as a GC box, and one or more copies of an 11-base pair (bp) conserved A+T-rich element, designated TAATGARAT. The TAATGARAT elements are thought to mediate the trans-activation of IE RNA synthesis by a virion-associated protein(s)6–14, and the flanking G + C-rich sequences appear both to potentiate this induction and to direct IE promoter activity in vivo9–14. The similarity of the herpesvirus GC box repeats to those of the simian virus 40 (SV40) early promoter15–17 prompted the in vitro analysis of HSV IE transcription reported here. We show that the mammalian gene-specific transcription factor Spl (refs 18–20) binds to eight distinct regions of the HSV short terminal repeat and stimulates transcription 25-fold from the divergent IE-3 (ICP-4) and IE-4/5 promoters.

Control of cytochrome P1-450 gene expression by dioxin.

Abstract: The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may produce its effects by altering gene expression in susceptible cells. In mouse hepatoma cells, TCDD induces the transcription of the cytochrome P1-450 gene, whose product, aryl hydrocarbon hydroxylase, contributes both to the detoxification and to the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons. A DNA fragment containing sequences flanking the 5' end of the cytochrome P1-450 gene was isolated and analyzed. This DNA fragment contains a cis-acting control element with at least three functional domains: a putative promoter, an inhibitory domain upstream from the promoter that blocks its function, and a TCDD-responsive domain still farther (1265 to 1535 base pairs) upstream of the promoter. These findings, together with results from earlier studies, imply that transcription of the cytochrome P1-450 gene is under both positive and negative control by at least two trans-acting regulatory factors.

E1A 13S and 12S mRNA products made in Escherichia coli both function as nucleus-localized transcription activators but do not directly bind DNA.

Abstract: We previously purified and characterized functionally the Escherichia coli-expressed product of the human subgroup C adenovirus E1A 13S mRNA (B. Ferguson, N. Jones, J. Richter, and M. Rosenberg, Science 224:1343-1346, 1984; B. Krippl, B. Ferguson, M. Rosenberg, and H. Westphal, Proc. Natl. Acad. Sci. USA 81:6988-6992, 1984). We have now expressed in E. coli and purified the protein product encoded by the human subgroup C adenovirus E1A 12S mRNA and have compared the functional properties of this protein with those of the E1A 13S mRNA product. Using microinjection techniques to introduce these proteins into mammalian cells, we found that the E1A 12S mRNA product, like the 13S mRNA product, localized rapidly to the cell nucleus and induced adenovirus gene expression. Although both E1A gene products localized to the nucleus and stimulated adenovirus gene transcription, these proteins did not directly bind to DNA under conditions in which a known DNA-binding protein, the human c-myc gene product, bound DNA efficiently. Thus, the E1A and myc gene products, which have been related both structurally and functionally, exhibit distinctly different biochemical properties.

Mechanisms of nucleolar dominance in animals and plants.

Abstract: In interspecific hybrids, it is often observed that the ribosomal genes of one species are transcriptionally dominant over the ribosomal genes of the other species. This phenomenon has been called "nucleolar dominance" and has been reported in such diverse organisms as frogs (Xenopus), Drosophila, many genera of plants, and mammalian somatic cell hybrids. Recent advances in our knowledge of the structure of ribosomal genes and their transcription machinery have led to proposals that at least two different molecular mechanisms can operate to cause nucleolar dominance and that the relative contribution of each mechanism is different for different types of crosses. One proposed mechanism involves competition between ribosomal genes which possess unequal numbers of enhancer elements. This mechanism can be abbreviated as the "enhancer imbalance mechanism." The second proposed mechanism involves the fact that the ribosomal gene (RNA polymerase I) transcription machinery evolves more rapidly between species than does the machinery for the other two classes of polymerase. This leads to dominance effects based on the apparent inability of a key transcription factor from one species to recognize the ribosomal gene promoter of the other species. This mechanism will be referred to as the "speciesspecific factor mechanism." The purpose of this review is to briefly summarize the evidence for these two molecular mechanisms and then to examine each of the known types of nucleolar dominance to assess how well the proposed mechanisms can account for each case.

Location of cis-acting regulatory sequences in the human T-cell leukemia virus type I long terminal repeat.

Abstract: The location of cis-acting regulatory regions within the long terminal repeat (LTR) of the human T-cell leukemia virus type I (HTLV-1) was determined. The sequences present between nucleotides -350 and -55 (cap site +1) contain an enhancer element that is active in lymphoid and nonlymphoid cell lines. The sequences located near the "TATA" and RNA initiation sites contain a promoter, the activity of which can be augmented by homologous and heterologous enhancer elements. A region responsive to trans-acting transcription factors present in HTLV-I- and HTLV type II-infected cells is located between nucleotides -159 and +315. HTLV-I LTR deletion mutants respond in a similar manner both to the trans-acting factors present in infected cells and to the tat protein encoded by the x-lor region of the genome, thus providing further evidence that the tat protein mediates transcriptional trans-activation of the LTR in HTLV-infected cells.

A trans-acting factor is responsible for the simian virus 40 enhancer activity in vitro

Abstract: Stimulation of in vitro transcription by the simian virus 40 enhancer involves a rapid and stable binding of a trans-acting factor with both the 5′- and 3′-domains of the enhancer sequence. The enhancer factor, which differs from other types of transcriptional factors, can interact with other enhancer elements.

Cell cycle control of the human HSP70 gene: implications for the role of a cellular E1A-like function.

Abstract: The gene encoding the human 70-kilodalton heat shock protein (HSP70) is subject to activation by the adenovirus E1A gene product and appears to be regulated in the absence of heat shock by a cellular activity similar to E1A. Given the relation of E1A to alteration of growth control, we have investigated the expression of the HSP70 gene during the cell cycle. Assay of mRNA levels after release from a thymidine-aphidicolin block revealed a 20-fold increase in mRNA abundance, reaching a peak level in the post-S-phase period. Upon reaching this peak level, the abundance of the mRNA then declined as the cells entered the next cycle. Control of the abundance of the mRNA during the cell cycle appeared to be primarily at the level of transcription as measured in nuclear runoff assays. Very similar results were obtained by analyzing the expression of the HSP70 gene in the adenovirus-transformed 293 cell line. Furthermore, the E1A gene was also found to be cell cycle regulated; the activation and peak level of the E1A mRNA occurred at an earlier time than those of the heat shock mRNA, consistent with, but not proof of, the hypothesis that E1A is responsible for the cell cycle control of the HSP70 expression. We therefore suggest that the E1A-like cellular activity may govern certain aspects of cell cycle transcription.

Effects of alterations in the 3′ flanking sequence on in vivo and in vitro expression of the yeast SUP4-o tRNATyr gene.

Abstract: The SUP4-o gene of Saccharomyces cerevisiae codes for an altered tRNATyr capable of suppressing ochre mutations. We constructed mutant SUP4-o genes with deletions in the 3'-flanking sequence and tested each for its ability to suppress ochre mutations in transformed yeast cells. The effects of the different 3' deletions on various aspects of in vitro transcription and RNA processing were also determined, using a yeast cell-free extract. Deletions that leave five or fewer consecutive T residues in the 3'-flanking sequence of SUP4-o were found to result in decreased efficiency of transcription termination, both in vitro and in vivo. Unexpectedly, the suppression strength of each mutant SUP4-o gene is highly correlated with the relative extent of transcription termination at the 3' end of the gene. This result indicates that SUP4-o readthrough transcripts are not efficiently processed to functional suppressor tRNA in yeast cells. Deletions that extend into the T cluster in the 3'-flanking sequence also significantly decrease the ability of SUP4-o to compete for a transcription factor that is limiting in our extracts. This latter finding implies that the 3'-flanking sequence of SUP4 plays a role in transcription factor binding.

Nitrogen regulation in Salmonella typhimurium. Identification of an ntrC protein-binding site and definition of a consensus binding sequence.

Abstract: We have investigated the DNA-binding ability of a nitrogen regulatory protein, the product of the ntrC gene, to several nitrogen-regulated promoters in Salmonella typhimurium. The ntrC protein is able to bind to the regulatory region (dhuA) of an operon coding for genes involved in the active transport of histidine, but not to another transport-related regulatory region, argTr. It bound to two different sites within the regulatory region of glnA (glutamine synthetase) and to one site in the regulatory region for the ntrBC operon. A consensus sequence has been derived from these four binding sites. The binding sequence displays dyad symmetry, as expected for the dimeric ntrC protein. The relationship of the binding sites to regulation of transcription initiation and termination, and to published homologies within the sequences of regulatory sites for nif genes is discussed.

trans Activation of the simian virus 40 late transcription unit by T-antigen.

Abstract: We have investigated the role of simian virus 40 (SV40) T-antigen in the induction of late gene expression independent of its function in amplifying templates through DNA replication. Northern blot and S1 nuclease analyses showed that stimulation occurred at the transcriptional level. At least two template elements, the T-antigen-binding sites and the 72-base-pair repeats, appeared to be important for this induction. Using template mutants, we demonstrated that deletions within T-antigen-binding site II decreased T-antigen-mediated late gene expression approximately 10- to 20-fold. In addition, multiple point mutations within a single retained copy of the SV40 72-base-pair repeat decreased T-antigen-mediated late gene expression. Using in vivo competition studies, we demonstrated that competitor DNA fragments containing the SV40 control region (nucleotides 5171 through 272) quantitatively decreased SV40 late gene expression in COS-1 cells. In contrast, competition with a plasmid containing SV40 nucleotides 1 through 294 (which removes all of T-antigen-binding site I and half of site II) was much less efficient. Finally, we demonstrated that in vivo competition experiments employing competitor fragments distal to the T-antigen-binding sites within the late template region (SV40 nucleotides 180 through 2533) resulted in superinduction of late gene expression in COS-1 cells. This finding suggests that negative factors such as repressors or attenuators may modulate late SV40 gene expression before induction. Our results are consistent with a model in which induction of late gene expression involves an interaction of the SV40 origin region with DNA-binding proteins, one of which may be T-antigen. Activation of the SV40 late transcription unit may involve induction of the SV40 enhancer or removal of a repressor-like protein or both.

A split binding site for transcription factor tau on the tRNA3Glu gene.

Abstract: Yeast transcription factor tau forms a stable complex with tRNA genes. Using this property, the factor could be highly purified on a specific tDNA column. The purified factor was found by DNA footprinting to protect the whole yeast tRNA3Glu gene from position -8 to +81. A DNase-sensitive site was retained in the middle of the gene on both strands. The 3' border of the complex was mapped by exonuclease digestion at +88, just downstream of the termination signal. The 5' limit of the complex was found at position -11. However, upon prolonged incubation with exonuclease, the -11 blockage disappeared and the DNA molecules were digested to position +30 to 38 in the middle of the gene. Contact points at guanine residues were identified by dimethyl sulphate protection experiments. Reduced methylation of G residues in the presence of factor was found solely within the A block and in the B block region. All six invariant GC pairs (i.e., G10, G18, G19 and G53, C56 and C61) were found to have strong contacts with the factor. These results show that tau factor interacts with both the 5' and 3' half of the tRNA3Glu gene, with the B block region being the predominant binding site. The presence of this dual binding site suggests a model in which the factor would bind alternately at the A and B block regions to allow transcription of the internal promoter by RNA polymerase C.

Transcription factor Sp1 recognizes promoter sequences from the monkey genome that are simian virus 40 promoter.

Abstract: A 440-base-pair fragment of African green monkey genomic DNA shares homology with the transcriptional regulatory region of simian virus 40 (SV40) and has been reported to direct transcription in vivo. We find that two regions within this fragment bind the promoter-specific cellular transcription factor Sp1 and are protected in DNase protection ("footprinting") experiments. As in SV40, binding occurs in regions containing multiple copies of the sequence GGGCGG. These regions, when fused to the proximal, or "TATA box," element of the herpes simplex virus thymidine kinase promoter, are able to direct Sp1-dependent transcription in vitro. The finding that Sp1 is capable of productive interaction with sequences taken from a cellular promoter supports the idea that Sp1 may play a role in modulating transcription of cellular genes.

The two embryonic U1 RNA genes of Xenopus laevis have both common and gene‐specific transcription signals.

Abstract: We have cloned and sequenced the 1842-bp repeat DNA encoding the two Xenopus laevis embryonic U1 RNAs, xU1a and xU1b. Although these two U1 RNAs are almost identical in sequence and are coordinately expressed during early embryogenesis, the flanking sequences of their genes show very little homology. Both genes contain two short conserved sequences, centered around positions -55 and +19, that probably are essential for 5' and 3' end formation of U1 RNAs, respectively. Efficient transcription of either gene in stage VI oocytes requires gene-specific promoter elements, located upstream of position -220. In the xU1b gene, these required 5'-flanking sequences include an 18-bp palindrome that has potential for Z-DNA formation. When injected separately into stage VI oocytes, the xU1a and xU1b genes are equally well transcribed, but co-injection of the two genes, either as the full length repeat or as two separate subclones, results in preferential accumulation of xU1b RNA. This competitive advantage of the xU1b gene in injected oocytes apparently is the result of preferred binding of one or more transcription factors that are limiting in these oocytes.

Characterization of the RNA binding properties of transcription factor IIIA of Xenopus laevis oocytes

Abstract: A nitrocellulose filter binding assay has been developed to study the interaction of Xenopus transcription factor IIIA with 5S RNA. The protein binds Xenopus oocyte 5S RNA with an association constant of 1.4 X 10(9) M-1 at 0.1 M salt, pH 7.5 at 20 degrees C. TF IIIA binds wheat germ 5S RNA with a two-fold higher affinity, E. coli 5S RNA with a four-fold weaker affinity, and has a barely detectable interaction with yeast tRNAphe. The preference for binding eukaryotic 5S RNA is enhanced in competition assays. The homologous reconstituted complex contains one molecule each of protein and 5S RNA and is indistinguishable from native 7S RNP in mobility on non-denaturing polyacrylamide gels. The conformation of the RNA in reconstituted particles is identical to the conformation of RNA in native 7S RNP. Further analysis of the homologous interaction reveals that complex formation is a favoured both by enthalpy and entropy. The 5S RNA binding activity has a broad pH optimum spanning pH 6.0 to pH 8.0. Determination of the salt dependence of Ka reveals that as many as 5 lysine-phosphate type ionic bonds may be formed in the homologous complex. Approximately 68% of the free energy of complex formation is contributed by non-electrostatic interactions between TF IIIA and Xenopus 5S RNA.

Transcription of vaccinia virus early genes by a template-dependent soluble extract of purified virions.

Abstract: An extract capable of selectively transcribing early vaccinia virus genes was prepared by disrupting purified vaccinia virions and passing the soluble material through a DEAE-cellulose column to remove endogenous DNA. Runoff transcripts of predicted size were synthesized by using double-stranded DNA templates that contained truncated early vaccinia virus genes, whereas several late vaccinia virus genes were not transcribed under these conditions. Proper dilution of the enzyme extract was critical, and a threshold concentration of DNA was required. At 30 degrees C, runoff transcripts were detected after 5 min and synthesis slowed appreciably after 30 min. Mg2+ was the preferred divalent cation, and KCl concentrations above 20 mM were inhibitory. Correct initiation of transcription was demonstrated by high-resolution analysis of S1 nuclease-digested hybrids formed by annealing in vitro-synthesized RNA with 5'-end-labeled DNA. A requirement for a 31-base-pair transcriptional regulatory sequence was found by using templates with deletions in an early promoter region. This in vitro system may be useful for mapping early transcriptional initiation sites, measuring the effects of additional promoter mutations, and isolating transcription factors.

A complex control region of the mouse rRNA gene directs accurate initiation by RNA polymerase I.

Abstract: To determine the size and location of the mouse rDNA promoter, we constructed systematic series of deletion mutants approaching the initiation site from the 5' and 3' directions. These templates were transcribed in vitro under various conditions with S-100 and whole-cell extracts. Surprisingly, the size of the rDNA region that determines the level of transcription differed markedly, depending on the reaction conditions. In both kinds of cell extracts, the apparent 5' border of the promoter was at residue ca. -27 under optimal transcription conditions, but as reaction conditions became less favorable, the 5' border moved progressively out to residues -35, -39, and -45. The complete promoter, however, extends considerably further, for under other nonoptimal conditions, we observed major effects of promoter domains extending in the 5' direction to positions ca. -100 and -140. In contrast, the apparent 3' border of the mouse rDNA promoter was at residue ca. +9 under all conditions examined. We also show that the subcloned rDNA region from -39 to +9 contains sufficient information to initiate accurately and that the region between +2 and +9 can influence the specificity of initiation. These data indicate that, although the polymerase I transcription factors recognize and accurately initiate with only the sequences downstream of residue -40, sequences extending out to residue -140 greatly favor the initiation reaction; presumably, this entire region is involved in rRNA transcription in vivo.

Autorepressor properties of the π-initiation protein encoded by plasmid R6K

Abstract: A DNA fusion containing the promoter of the pir gene of plasmid R6K that encodes for the pi-initiation protein and the beta-galactosidase gene of Escherichia coli (lacZ) is described. The synthesis of beta-galactosidase promoted by this pir-lac fusion was almost completely inhibited when an R6K sequence containing the pir gene was provided in trans in E. coli. Transcription in vitro from the pir promoter but not the trp promoter of E. coli, was inhibited by purified pi protein indicating that the pi protein alone is responsible for repression of its own gene and that the effect is promoter specific. The DNA-protein interaction sites in the pir regulatory region have been determined for the pi protein and E. coli RNA polymerase using the DNase I protection method. The binding sites for these two proteins overlap for three helical turns. Competition DNA binding experiments show that the pi protein will displace bound RNA polymerase. From these studies we conclude that repression of the pir gene is accomplished by binding of the pi protein and this association blocks access of RNA polymerase to the pir promoter region.

The relationship of regulatory proteins and DNase I hypersensitive sites in the yeast GAL1-10 genes

Abstract: We have used yeast strains containing a disrupted positive (GAL4) and/or a disrupted negative (GAL80) regulatory gene to investigate the relationship of these regulatory proteins to the hypersensitive sites upstream of their target genes, GAL1-10. We find that neither of these regulatory proteins is required for the formation of the hypersensitive region. There is positive regulatory protein (dependent) binding to a portion of the hypersensitive region when GAL1 and 10 are expressed. However, similar binding can also occur under conditions in which the genes are not expressed. Thus, such binding is necessary but not sufficient for expression of GAL1 and 10 and control of GAL1-10 expression must also include processes which occur subsequent to GAL4/DNA binding. The negative regulatory protein GAL80 plays a significant role in these processes.

Transcriptional analysis of the adenovirus-5 EIII promoter: absence of sequence specificity for stimulation by EIa gene products.

Abstract: To identify the adenovirus-5 EIII promoter sequences that are involved in basal level of transcription, a series of promoter deletion mutants were analyzed in vivo by transfection into HeLa cells and in vitro using a HeLa whole cell extract system. Three regions within the EIII promoter were shown to be important for efficient transcription: the TATA sequence, an upstream element centered at -55/-57, and an additional element located between -111 and -233. In vivo transcriptional analysis of EIII promoter deletions in the presence of adenovirus EIa gene products have demonstrated that the same three regions are required for EIa-stimulated transcription. We conclude that there is no sequence element in the EIII promoter between -15 and -233 that is uniquely required for the stimulation of EIII transcription by EIa gene products.