ABSTRACT
TFIIA interacts with TFIID via association with TATA binding protein (TBP) and TBP-associated factor 11 (TAF11). We previously identified a mutation in the small subunit of TFIIA (toa2-I27K) that is defective for interaction with TAF11. To further explore the functional link between TFIIA and TAF11, the toa2-I27K allele was utilized in a genetic screen to isolate compensatory mutants in TAF11. Analysis of these compensatory mutants revealed that the interaction between TAF11 and TFIIA involves two distinct regions of TAF11: the highly conserved histone fold domain and the N-terminal region. Cells expressing a TAF11 allele defective for interaction with TFIIA exhibit conditional growth phenotypes and defects in transcription. Moreover, TAF11 imparts changes to both TFIIA-DNA and TBP-DNA contacts in the context of promoter DNA. These alterations appear to enhance the formation and stabilization of the TFIIA-TBP-DNA complex. Taken together, these studies provide essential information regarding the molecular organization of the TAF11-TFIIA interaction and define a mechanistic role for this association in the regulation of gene expression in vivo.
Subject(s)
Models, Molecular , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , TATA-Binding Protein Associated Factors/metabolism , TATA-Box Binding Protein/metabolism , Transcription Factor TFIIA/metabolism , Transcription Factor TFIID/metabolism , Gene Expression Regulation, Fungal/genetics , Mutation , Promoter Regions, Genetic/genetics , Protein Binding , Protein Structure, Tertiary/physiologyABSTRACT
To determine the mechanistic differences between canonical and noncanonical TATA elements, we compared the functional activity of two sequences: TATAAA (canonical) and CATAAA (noncanonical). The TATAAA element can support high levels of transcription in vivo, whereas the CATAAA element is severely defective for this function. This dramatic functional difference is not likely to be due to a difference in TBP (TATA-binding protein) binding efficiency because protein-DNA complex studies in vitro indicate little difference between the two DNA sequences in the formation and stability of the TBP-DNA complex. In addition, the binding and stability of the TFIIB-TBP-DNA complex is similar for the two elements. In striking contrast, the TFIIA-TBP-DNA complex is significantly less stable on the CATAAA element when compared with the TATAAA element. A role for TFIIA in distinguishing between TATAAA and CATAAA in vivo was tested by fusing a subunit of TFIIA to TBP. We found that fusion of TFIIA to TBP dramatically increases transcription from CATAAA in yeast cells. Taken together, these results indicate that the stability of the TFIIA-TBP complex depends strongly on the sequence of the core promoter element and that the TFIIA-TBP complex plays an important function in recognizing optimal promoters in vivo.
Subject(s)
DNA-Binding Proteins/chemistry , DNA/metabolism , Transcription Factors/chemistry , DNA-Binding Proteins/genetics , Kinetics , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , Recombinant Proteins/metabolism , Repetitive Sequences, Nucleic Acid , TATA-Box Binding Protein , Time Factors , Transcription Factor TFIIA , Transcription Factors/genetics , Transcription, Genetic , Yeasts/chemistry , Yeasts/geneticsABSTRACT
TFIIA and TATA-binding protein (TBP) associate directly at the TATA element of genes transcribed by RNA polymerase II. In vivo, TBP is complexed with approximately 14 TBP-associated factors (TAFs) to form the general transcription factor TFIID. How TFIIA and TFIID communicate is not well understood. We show that in addition to making direct contacts with TBP, yeast TAF40 interacts directly and specifically with TFIIA. Mutational analyses of the Toa2 subunit of TFIIA indicate that loss of functional interaction between TFIIA and TAF40 results in conditional growth phenotypes and defects in transcription. These results demonstrate that the TFIIA-TAF40 interaction is important in vivo and indicate a functional role for TAF40 as a bridging factor between TFIIA and TFIID.
Subject(s)
DNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins , TATA-Binding Protein Associated Factors , Transcription Factors, TFII/metabolism , Transcription Factors/metabolism , Cell Survival , DNA Mutational Analysis , DNA-Binding Proteins/chemistry , Dose-Response Relationship, Drug , Escherichia coli/metabolism , Fungal Proteins/metabolism , Glutathione Transferase/metabolism , Models, Molecular , Mutagenesis , Mutation , Phenotype , Protein Binding , Protein Structure, Tertiary , TATA-Box Binding Protein , Temperature , Transcription Factor TFIIA , Transcription Factor TFIID , Transcription Factors/chemistry , Transcription Factors, TFII/chemistry , Transcription, Genetic , Transcriptional Activation , Two-Hybrid System TechniquesABSTRACT
TFIIA contributes to transcription initiation by stabilizing the TBP-TATA interaction and by mediating the response to transcriptional activators and inhibitors. TFIIA contains a six-stranded beta-sheet domain and a four-helix bundle. The beta-domain makes functional contacts with DNA and TBP. The role of the four-helix bundle was investigated using a structure-based model of this domain (called 4HB). 4HB adopts a highly stable, helical fold, consistent with its structure in the context of TFIIA. Like TBP and other intact transcription factors, 4HB is able to activate transcription in vivo when artificially recruited to a promoter via a heterologous DNA-binding domain. Thus, in addition to making important contacts with TBP and DNA via the beta-domain, TFIIA makes other specific, functional contacts with the transcriptional machinery via the four-helix bundle. Proteins 2001;43:227-232.
Subject(s)
DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Saccharomyces cerevisiae Proteins , TATA-Binding Protein Associated Factors , Transcription Factors/genetics , Transcriptional Activation , Animals , Circular Dichroism , DNA-Binding Proteins/chemistry , Fungal Proteins/chemistry , Models, Molecular , Protein Conformation , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Spectrum Analysis , Transcription Factor TFIID , Transcription Factors/chemistry , Transcription, GeneticABSTRACT
The physiological role of TFIIA was investigated by analyzing transcription in a yeast strain that contains a TATA-binding protein (TBP) mutant (N2-1) defective for interacting with TFIIA. In cells containing N2-1, transcription from a set of artificial his3 promoters dependent on different activators is generally reduced by a similar extent, indicating that TFIIA function is largely nonselective for activators. In addition, TATA element utilization, a core promoter function, is altered at his3 promoters dependent on weak activators. Genomic expression analysis reveals that 3% of the genes are preferentially affected by a factor of 4 or more. Chimeras of affected promoters indicate that the sensitivity to the TFIIA-TBP interaction can map either to the upstream or core promoter region. Unlike wild-type TBP or TFIIA, the N2-1 derivative does not activate transcription when artificially recruited to the promoter via a heterologous DNA binding domain, indicating that TFIIA is important for transcription even in the absence of an activation domain. Taken together, these results suggest that TFIIA plays an important role in both activator-dependent and core promoter functions in vivo. Further, they suggest that TFIIA function may not be strictly related to the recruitment of TBP to promoters but may also involve a step after TBP recruitment.
Subject(s)
Promoter Regions, Genetic , Saccharomyces cerevisiae Proteins , Transcription Factors/physiology , Yeasts/genetics , Bacterial Proteins/genetics , Binding Sites , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Genes, Fungal , Hydro-Lyases/genetics , Kinetics , Mutation , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Recombinant Fusion Proteins/genetics , Serine Endopeptidases/genetics , TATA-Box Binding Protein , Transcription Factor TFIIA , Transcription Factors/genetics , Transcription, GeneticABSTRACT
The TATA-binding protein (TBP) plays an important role in transcriptional initiation by all three nuclear RNA polymerases. TBP contains a conserved C-terminal domain (cTBP) that binds DNA. Crystallographic studies of cTBP (i.e., TBP without the N-terminal domain) from various species and molecular biology studies of cTBP and mixed cTBP/TBP species have led to the view that DNA binding by TBP is regulated by TBP dimerization. Using sedimentation equilibrium, we show that yeast cTBP forms dimers in solution at 5 degrees C with a dissociation constant of 7 +/- 1 microM. This observation of cTBP dimers in solution is in accord with the dimeric state observed in crystal structures of cTBP. In contrast, physiologically relevant, full-length yeast TBP is monomeric at 5 degrees C and forms dimers at 30 degrees C with a dissociation constant of 51 +/- 16 microM. This dissociation constant precludes formation of stable full-length TBP dimers at physiological concentrations. In addition, we tested for yeast TBP oligomerization in the presence of TBP-associated factors in the context of TFIID. No evidence for TBP oligomers was found using immunoprecipitation techniques from yeast whole-cell extracts. We conclude that yeast TBP is predominantly monomeric under physiological conditions, arguing against a role for TBP dimerization in the regulation of transcriptional initiation.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Polymers/chemistry , Polymers/metabolism , TATA Box , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription, Genetic , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Dimerization , Escherichia coli/genetics , Kinetics , Molecular Weight , Osmolar Concentration , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Saccharomyces cerevisiae/genetics , TATA Box/genetics , TATA-Box Binding Protein , Transcription Factors/biosynthesis , Transcription Factors/genetics , UltracentrifugationABSTRACT
The genetic makeup of animal and plant populations is determined by established principles and concepts. Ecology and evolution provide a basic theoretical framework for understanding how genetic changes occur in populations. Whether these rules can be applied to host retroviral populations is unknown. Individuals infected with the human immunodeficiency virus (HIV) contain within their bodies a viral population. This population is known as a viral quasispecies. Located in the transmembrane protein of HIV-1 is the viral sequence Gly-Thr-Asp-Arg-Val. Previous immunological studies have shown that viral antibody is produced in response to this five-amino-acid sequence. Antibody to this viral sequence also crossreacts and binds to a related peptide sequence found on certain immune cells. This related sequence, Gly-Thr-Glu-Arg-Val, is found on immune cells bearing a structure known as the major histocompatibility complex (MHC). The viral transmembrane sequence, Gly-Thr-Asp-Arg-Val, can be substituted with alanine residues utilizing site-directed mutagenesis. This creates a viral clone devoid of the genetic similarity with the MHC. Chimpanzees progressing to AIDS contain both sequences of interest. Suppression of the chimpanzee quasispecies utilizing anti-retroviral drugs is proposed. This action serves to suppress the presence of the viruses containing the sequence Gly-Thr-Asp-Arg-Val. When viral load has been reduced significantly, a drug resistant, alanine altered clone is to be introduced in large numbers. The concept of evolutionary stable strategy predicts that a viable HIV clone with alanine residues can genetically dominate the viral population. Immune system recognition of the alanine sequence is likely to result in renewed antibody production. Antibodies to the alanine containing viral sequence should not recognize or bind to the MHC. Immunological parameters can then be measured to determine the physiological impact of eliminating a sequence responsible for molecular mimicry between virus and host.
Subject(s)
Acquired Immunodeficiency Syndrome/virology , HIV-1/immunology , Molecular Mimicry/genetics , Acquired Immunodeficiency Syndrome/genetics , Acquired Immunodeficiency Syndrome/immunology , Animals , Ape Diseases/genetics , Ape Diseases/immunology , Ape Diseases/virology , Genes, MHC Class II/genetics , HIV-1/genetics , Humans , Immune System/immunology , Immune System/virology , Polymorphism, Genetic , Sequence HomologyABSTRACT
Using an intragenic complementation screen, we have identified a temperature-sensitive TATA-binding protein (TBP) mutant (K151L, K156Y) that is defective for interaction with certain yeast TBP-associated factors (TAFs) at the restrictive temperature. The K151L,K156Y mutant appears to be functional for RNA polymerase I (Pol I) and Pol III transcription, and it is capable of supporting Gal4-activated and Gcn4-activated transcription by Pol II. However, transcription from certain TATA-containing and TATA-less Pol II promoters is reduced at the restrictive temperature. Immunoprecipitation analysis of extracts prepared after culturing cells at the restrictive temperature for 1 h indicates that the K151L,K156Y derivative is severely compromised in its ability to interact with TAF130, TAF90, TAF68/61, and TAF25 while remaining functional for interaction with TAF60 and TAF30. Thus, a TBP mutant that is compromised in its ability to form TFIID can support the response to Gcn4 but is defective for transcription from specific promoters in vivo.
Subject(s)
TATA Box/physiology , Transcription Factors, TFII/physiology , Amitrole/metabolism , DNA Polymerase II/physiology , DNA Polymerase III/physiology , Enzyme Inhibitors/metabolism , Immunoblotting , Models, Molecular , Mutagenesis , Phenotype , Precipitin Tests , Saccharomyces cerevisiae/genetics , Temperature , Transcription Factor TFIID , Transcription, GeneticABSTRACT
The oncoprotein Tax, encoded by the human T-cell leukemia virus type I (HTLV-I), is required for high-level viral transcription and is strongly linked to HTLV-I-associated malignant transformation. Recent evidence suggests that Tax stimulates HTLV-I transcription through recruitment of the cellular coactivator protein CBP to the HTLV-I promoter, promoting high-level viral replication via the transcriptional activation properties associated with CBP. Tax directly contacts the KIX domain of CBP to stably anchor the coactivator to nucleoprotein complexes at the promoter. Here, we identify KIX amino acid residues 588 to 683 as the minimal region sufficient for interaction with Tax. This region is similar to the minimal KIX amino acid residues necessary for strong interaction with phosphorylated CREB, and is composed of a structural domain that forms an extensive hydrophobic core. We further show that a double point mutation in KIX differentially affects the binding of Tax and phosphorylated CREB, suggesting that these transcription factors may recognize unique amino acid residues within the KIX domain. These observations suggest that Tax directly contacts the hydrophobic core of KIX, and provides a structural framework to further define the molecular interactions between Tax and CBP.
Subject(s)
Gene Products, tax/metabolism , Human T-lymphotropic virus 1/metabolism , Nuclear Proteins/metabolism , Trans-Activators/metabolism , CREB-Binding Protein , Humans , Nuclear Proteins/genetics , Phosphorylation , Point Mutation , Protein Binding , Recombinant Fusion Proteins , Sequence Deletion , Trans-Activators/genetics , Transcriptional Activation/physiologyABSTRACT
Transcriptional activation involves the regulated assembly of multiprotein complexes on promoter DNA in the context of the repressive effects of chromatin. How do activators orchestrate this complicated phenomenon in vivo? Recent genetic and biochemical advancements suggest that activator-dependent formation of the transcription machinery on the promoter involves at least two steps. First, the activator facilitates the recruitment of TFIID to the TATA element of the promoter. TFIID binding is then followed by the recruitment of the remainder of the transcriptional apparatus in the form of the RNA polymerase II holoenzyme.
Subject(s)
Saccharomyces cerevisiae/genetics , TATA Box , Transcription Factors/metabolism , Transcriptional Activation , Chromatin/physiology , Models, Genetic , Promoter Regions, Genetic , Protein Binding , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factor TFIIA , Transcription Factor TFIIB , Transcription Factor TFIID , Transcription, GeneticABSTRACT
Using a genetic screen, we isolated four TATA-binding protein (TBP) mutants that are specifically defective in vivo for the response to acidic activators. In contrast to previously described activation-defective TBP mutants, these TBP derivatives are not specifically defective for interactions with TATA elements or TFIIA. Three of these derivatives interact normally with a TATA element, TFIIA, TFIIB, or an acidic activation domain; presumably, they affect another protein-protein interaction important for transcriptional activation. The remaining derivative (with F-237 replaced by D) binds a TATA element with wild-type affinity, but the TBP-TATA complex has an altered electrophoretic mobility and interacts poorly with TFIIA and TFIIB; this suggests that the conformation of the TBP-TATA element complex plays a role in transcriptional activation. To determine the step at which the TBP derivatives were unable to activate transcription, we utilized an artificial recruitment assay in which TBP is targeted to the promoter via fusion to the LexA DNA-binding domain. Consistent with previous evidence that acidic activators can increase recruitment of TBP to the promoter in vivo, the activation defect of some of these TBP derivatives can be corrected by artificial recruitment. In contrast, the activation defect of the other TBP derivatives is not bypassed by artificial recruitment. Thus, these TBP mutants define two steps in the process of transcriptional stimulation by acidic activators: efficient recruitment to the TATA element and a postrecruitment interaction with a component(s) of the initiation complex.
Subject(s)
DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Transcription Factors/physiology , Transcription, Genetic , Transcriptional Activation , DNA-Binding Proteins/chemistry , Genetic Complementation Test , Macromolecular Substances , Promoter Regions, Genetic , Protein Conformation , RNA Polymerase III/metabolism , Saccharomyces cerevisiae , Signal Transduction , Structure-Activity Relationship , TATA-Box Binding Protein , Transcription Factor TFIIA , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
Mammalian cells regulate tubulin mRNA abundance by a posttranscriptional mechanism dependent on the concentration of tubulin monomer. Treatment of mammalian cells with microtubule-depolymerizing drugs and microtubule-polymerizing drugs causes decreases and increases in tubulin mRNA, respectively (D. W. Cleveland, Curr. Opin. Cell Biol. 1:10-14, 1989). In striking contrast to the case with mammalian cells, perturbation of microtubules in Tetrahymena thermophila by microtubule-depolymerizing or -polymerizing drugs increases the level of the single alpha-tubulin gene message by increasing transcription (L. A. Stargell, D. P. Heruth, J. Gaertig, and M. A. Gorovsky, Mol. Cell. Biol. 12:1443-1450, 1992). In this report we show that antimicrotubule drugs preferentially induce the expression of one of two beta-tubulin genes (BTU1) in T. thermophila. In contrast, deciliation induces expression of both beta-tubulin genes. Tubulin gene expression was examined in a mutant strain created by transformation with an in vitro-mutagenized beta-tubulin gene that conferred resistance to microtubule-depolymerizing drugs and sensitivity to the polymerizing drug taxol and in a strain containing a nitrosoguanidine-induced mutation in the single alpha-tubulin gene that conferred the same pattern of drug sensitivities. In both cases the levels of tubulin mRNA expression from the drug-inducible BTU1 gene in the mutant cells paralleled the altered growth sensitivities to microtubule drugs. These studies demonstrate that T. thermophila has distinct, gene-specific mechanisms for modulating tubulin gene expression depending on whether ciliary or cytoplasmic microtubules are involved. They also show that the cytoplasmic microtubule cytoskeleton itself participates in a signal transduction pathway that regulates specific tubulin gene transcription in T. thermophila.
Subject(s)
Genes, Protozoan/genetics , Microtubules/metabolism , Signal Transduction , Sulfanilamides , Tetrahymena thermophila/physiology , Tubulin/genetics , Animals , Cilia/physiology , Dinitrobenzenes/pharmacology , Dose-Response Relationship, Drug , Gene Expression Regulation , Paclitaxel/pharmacology , RNA, Messenger/biosynthesis , Tetrahymena thermophila/drug effects , Transcription, GeneticABSTRACT
A yeast TBP mutant (N2-1) is described here that is defective specifically in responding to acidic activators in vivo. N2-1 does not support activation by Gal4, Ace1, and Gcn4, but appears unaffected for constitutive transcription, repression by the Cyc8-Tup1 and Not complexes, and transcription by polymerase I (Pol) and Pol III. In vitro, N2-1 fails to interact with TFIIA, but it associates normally with a TATA element, an acidic activation domain, and TFIIB. Fusion of the small subunit of TFIIA to N2-1 restores activation function in vivo. Thus, an efficient interaction between TBP and TFIIA is required for transcriptional activation in vivo.
Subject(s)
DNA-Binding Proteins/metabolism , Nuclear Proteins , Repressor Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , TATA Box , Trans-Activators/physiology , Transcription Factors/metabolism , Transcriptional Activation , Base Sequence , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , DNA-Directed RNA Polymerases/metabolism , Fungal Proteins/physiology , Hydrogen-Ion Concentration , Immediate-Early Proteins/physiology , Molecular Sequence Data , Mutation , Nuclear Receptor Subfamily 4, Group A, Member 2 , Protein Kinases/physiology , Saccharomyces cerevisiae/growth & development , TATA-Box Binding Protein , Transcription Factor TFIIA , Transcription Factors/genetics , Transcription Factors/physiologyABSTRACT
Although the TATA-binding protein (TBP) is highly conserved throughout the eukaryotic kingdom, human TBP cannot functionally replace yeast TBP for cell viability. To investigate the basis of this species specificity, we examine the in vivo transcriptional activity of human TBP at different classes of yeast promoters. Consistent with previous results, analysis of yeast/human hybrid TBPs indicates that growth defects are not correlated with the ability to promote TATA-dependent polymerase II (Pol II) transcription or to respond to acidic activator proteins. Human TBP partially complements the growth defects of a yeast TBP mutant with altered TATA element-binding specificity, suggesting that it carries out sufficient Pol II function to support viability. However, human TBP does not complement the defects of yeast TBP mutants that are specifically defective in transcription by RNA polymerase III. Three independently isolated derivatives of human TBP that permit yeast cell growth replace arginine 231 with lysine; the corresponding amino acid in yeast TBP (lysine 133) has been implicated in RNA polymerase III transcription. Transcriptional analysis indicates that human TBP functions poorly at promoters recognized by RNA polymerases I and III and at RNA Pol II promoters lacking a conventional TATA element. These observations suggest that species specificity of TBP primarily reflects evolutionarily diverged interactions with TBP-associated factors (TAFs) that are necessary for recruitment to promoters lacking TATA elements.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Directed RNA Polymerases/genetics , Promoter Regions, Genetic/genetics , Saccharomyces cerevisiae/growth & development , Transcription Factors/genetics , Transcription, Genetic/genetics , Amino Acid Sequence , Base Sequence , DNA-Directed RNA Polymerases/metabolism , Humans , Molecular Sequence Data , Mutation , Saccharomyces cerevisiae/genetics , Species Specificity , TATA Box , TATA-Box Binding ProteinABSTRACT
Unambiguous TATA boxes have not been identified in upstream sequences of Tetrahymena thermophila genes analyzed to date. To begin a characterization of the promoter requirements for RNA polymerase II, the gene encoding TATA-binding protein (TBP) was cloned from this species. The derived amino acid sequence for the conserved C-terminal domain of Tetrahymena TBP is one of the most divergent described and includes a unique 20-amino-acid C-terminal extension. Polyclonal antibodies generated against a fragment of Tetrahymena TBP recognize a 36-kDa protein in macronuclear preparations and also cross-react with yeast and human TBPs. Immunocytochemistry was used to examine the nuclear localization of TBP during growth, starvation, and conjugation (the sexual phase of the life cycle). The transcriptionally active macronuclei stained at all stages of the life cycle. The transcriptionally inert micronuclei did not stain during growth or starvation but surprisingly stained with anti-TBP throughout early stages of conjugation. Anti-TBP staining disappeared from developing micronuclei late in conjugation, corresponding to the onset of transcription in developing macronuclei. Since micronuclei do not enlarge or divide at this time, loss of TBP appears to be an active process. Thus, the transcriptional differences between macro- and micronuclei that arise during conjugation are associated with the loss of a major component of the basal transcription apparatus from developing micronuclei rather than its appearance in developing macronuclei.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Tetrahymena thermophila/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Base Sequence , Biological Evolution , Cell Nucleus/metabolism , Cloning, Molecular , DNA, Protozoan/genetics , DNA-Binding Proteins/immunology , Genes, Protozoan , Humans , Immunochemistry , Micronucleus, Germline/metabolism , Molecular Sequence Data , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Protozoan Proteins/metabolism , Sequence Homology, Amino Acid , TATA-Box Binding Protein , Tetrahymena thermophila/growth & development , Tetrahymena thermophila/metabolism , Transcription Factors/immunology , Transcription, GeneticABSTRACT
Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Although structurally and functionally dissimilar, these nuclei are products of a single postzygotic division during conjugation, the sexual phase of the life cycle. Immunocytochemical analyses during growth, starvation, and conjugation were used to examine the nuclear deposition of hv1, a histone H2A variant that is found in macronuclei and thought to play a role in transcriptionally active chromatin. Polyclonal antisera were generated using whole hv1 protein and synthetic peptides from the amino and carboxyl domains of hv1. The transcriptionally active macronuclei stained at all stages of the life cycle. Micronuclei did not stain during growth or starvation but stained with two of the sera during early stages of conjugation, preceding the stage when micronuclei become transcriptionally active. Immunoblot analyses of fractionated macro- and micronuclei confirmed the micronuclear acquisition of hv1 early in conjugation. hv1 staining disappeared from developing micronuclei late in conjugation. Interestingly, the carboxy-peptide antiserum stained micronuclei only briefly, late in development. The detection of the previously sequestered carboxyl terminus of hv1 may be related to the elimination of hv1 during the dynamic restructing of micronuclear chromatin that occurs as the micronucleus enters a transcriptionally incompetent state that is maintained during vegetative growth. These studies demonstrate that the transcriptional differences between macro- and micronuclei are associated with the loss of a chromatin component from developing micronuclei rather than its de novo appearance in developing macronuclei and argue that hv1 functions in establishing a transcriptionally competent state of chromatin.
Subject(s)
Cell Nucleus/chemistry , Histones/analysis , Protozoan Proteins/analysis , Tetrahymena thermophila/chemistry , Amino Acid Sequence , Animals , Chromatin/physiology , Chromatography, High Pressure Liquid , Fluorescent Antibody Technique , Histones/physiology , Immune Sera , Molecular Sequence Data , Protozoan Proteins/metabolism , Tetrahymena thermophila/genetics , Tetrahymena thermophila/growth & development , Transcription, Genetic/physiologyABSTRACT
In cultured mammalian cells, an increase in the amount of tubulin monomer due to treatment with a microtubule-depolymerizing agent results in a rapid decline in tubulin synthesis. This autoregulatory response is mediated through a posttranscriptional mechanism which decreases the stability of tubulin message with no change in transcriptional activity of tubulin genes. Conversely, treatment with a microtubule-polymerizing drug, such as taxol, results in a slight increase in the synthesis of tubulin. Surprisingly, we find that two microtubule-depolymerizing agents, colchicine and oryzalin, actually cause an increase in alpha-tubulin synthesis and alpha-tubulin message in starved Tetrahymena thermophila. This increase is paralleled by an increase in transcription of alpha-tubulin sequences measured by run-on transcription, while the half-life of tubulin message measured by decay in the presence of actinomycin D does not change appreciably. Treatment of starved cells with taxol also produces an increase in alpha-tubulin synthesis via an increase in message abundance due to an increase in transcription of the alpha-tubulin gene. These results indicate that tubulin synthesis in T. thermophila is regulated very differently than in cultured mammalian cells.
Subject(s)
Microtubules/metabolism , RNA, Messenger/metabolism , Sulfanilamides , Tetrahymena thermophila/metabolism , Transcription, Genetic/drug effects , Tubulin/metabolism , Alkaloids/pharmacology , Animals , Colchicine/pharmacology , Cytochalasin B/pharmacology , Dinitrobenzenes/pharmacology , Dose-Response Relationship, Drug , Food Deprivation , Immunohistochemistry , Microtubules/drug effects , Paclitaxel , RNA, Messenger/drug effects , Tetrahymena thermophila/drug effects , Tubulin/drug effects , Verapamil/pharmacologyABSTRACT
Conjugation in Tetrahymena represents an ordered developmental pathway which represents the sexual phase of the ciliate life cycle. This pathway is initiated when starved cells of opposite mating types are mixed and are allowed to make a series of cell-cell contacts (a period termed costimulation) which lead to the formation of mating pairs. Here, we demonstrate that two previously described abundant high mobility group (HMG)-like proteins, HMG B and HMG C, whose synthesis appeared to be coordinately regulated in vegetative cells, are not required during the same stages of conjugation. The level of mRNA for both HMG B and HMG C is high during vegetative growth and during the development of new macronuclei. However, specific induction of HMG B mRNA is observed soon after cells of opposite mating types are mixed. Thus, the genes which encode HMG B and HMG C in Tetrahymena can be controlled independently or coordinately. Nuclear run-on experiments show that a significant factor underlying the rapid induction of HMG B message early in the sexual cycle is an increase in the transcriptional activity of the HMG B gene. Experiments are presented which show that this induction of HMG B message requires protein synthesis and is dependent upon the cell-cell contacts made during costimulation. Essentially all of the HMG B protein, which is newly synthesized during this period, is targeted to parental macronuclei where it serves an as yet undetermined function(s).
Subject(s)
Cell Communication , High Mobility Group Proteins/physiology , Tetrahymena/physiology , Animals , Blotting, Northern , Cycloheximide/pharmacology , Gene Expression Regulation/drug effects , Molecular Weight , RNA, Messenger/genetics , Time Factors , Transcription, GeneticABSTRACT
The only well-characterized study of gene expression in Tetrahymena thermophila (1) demonstrates that the temperature dependent expression of the Ser H3 gene is regulated at the level of mRNA stability. A run-on transcription assay was developed to determine if regulation of RNA stability was a major mechanism regulating gene expression in Tetrahymena or if transcriptional regulation dominates. The relative transcriptional activities of 14 Tetrahymena genes were determined in different physiological/developmental states (growing, starved and conjugating) in which many of the genes showed striking differences in RNA abundance. In every case except Ser H3, changes in transcription accompanied changes in RNA abundance. Thus differential transcription, not differential RNA degradation, is the major mechanism regulating RNA abundance in Tetrahymena.