PubMed Health⌕ Search

Biomedical subjects

J A Jaehning

Publications and source records attributed to J A Jaehning.

At least 19 recordsLinked to original sources
PubMed1994

Release of the yeast mitochondrial RNA polymerase specificity factor from transcription complexes.

The yeast mitochondrial RNA polymerase is composed of two nuclear encoded subunits, a catalytic core (Rpo41p), which resembles the enzymes from bacteriophage T7 and T3, and a specificity factor required for promoter recognition (Mtf1p), which is similar to members of the eubacterial sigma factor family. Using mitochondrial RNA polymerase reconstituted from highly purified subunits, we have determined that Rpo41p and Mtf1p interact to form a holoenzyme in solution prior to DNA binding and promoter recognition. We analyzed the composition of the polymerase during and after the initiation of transcription and found that, like the eubacterial sigma factors, Mtf1p is released after initiation and is available to catalyze transcription on a second template. By analyzing gel mobility shift complexes of the RNA polymerase and DNA at different stages of the transcription reaction, we found that both subunits were associated with DNA prior to initiation and after the formation of two phosphodiester bonds. After the formation of a 13-nucleotide transcript, Mtf1p is no longer associated with Rpo41p on the DNA. These data establish that Mtf1p is functionally as well as structurally similar to eubacterial sigma factors.

Base Sequence↗
PubMed1994

Isolation of yeast transcription factor IIA using a functional transcription assay.

TFIIA was extensively purified from a whole-cell transcription extract from yeast. Activity was followed throughout isolation utilizing a functional transcription assay. Transcription activity was found to copurify with polypeptides of 43 and 12.5 kDa, consistent with a previous purification that utilized a TBP/DNA gel mobility shift assay (J. Ranish and S. Hahn, J. Biol. Chem. 266, 19320-19327, 1991). The Stoke's radius of the purified protein was determined by gel filtration chromatography to be 44 A under native conditions. The solution molecular weight derived from this measurement, 110 kDa, is consistent with a heterotetrameric structure of TFIIA.

Chromatography, Gel↗
PubMed1992

In vitro transcriptional activation by a metabolic intermediate: activation by Leu3 depends on alpha-isopropylmalate.

In the absence of the leucine biosynthetic precursor alpha-isopropylmalate (alpha-IPM), the yeast LEU3 protein (Leu3p) binds DNA and acts as a transcriptional repressor in an in vitro extract. Addition of alpha-IPM resulted in a dramatic increase in Leu3p-dependent transcription. The presence of alpha-IPM was also required for Leu3p to compete effectively with another transcriptional activator, GAL4/VP16, for limiting transcription factors. Therefore, the addition of alpha-IPM appears to convert a transcriptional repressor into an activator. This represents an example in eukaryotes of direct transcriptional regulation by a small effector molecule.

Binding, Competitive↗
PubMed1992

A transcriptionally active form of GAL4 is phosphorylated and associated with GAL80.

The GAL4 activator and GAL80 repressor proteins regulate the expression of yeast genes in response to galactose. A complex of the two proteins isolated from glucose-grown cells is inactive in an in vitro transcription reaction but binds DNA and blocks activation by the GAL4-VP16 chimeric activator. The complex purified from galactose-grown cells contains a mixture of phosphorylated and unphosphorylated forms of GAL4. The galactose-induced form of GAL4 activates in vitro transcription to levels similar to those seen with GAL4-VP16. The induced GAL4 complex is indistinguishable in size and apparent shape from the uninduced complex, consistent with a continued association with GAL80. These results confirm in vivo analyses that correlate GAL4 phosphorylation with galactose induction and support a model of transcriptional activation that does not require GAL80 dissociation.

Base Sequence↗
PubMed1992

The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding.

A variety of techniques, including filter binding, footprinting, and gel retardation, can be used to assay the transcriptional activator GAL4 (Gal4p) through the initial steps of its purification from yeast cells. Following DNA affinity chromatography, Gal4p still bound DNA selectively when assayed by filter binding or footprinting. However, the affinity-purified protein was no longer capable of forming a stable complex with DNA, as assayed by gel retardation. Mixing the purified Gal4p with the flowthrough fraction from the DNA affinity column restored gel retardation complex formation. Gel retardation assays were used to monitor the purification of a heat-stable Gal4p-DNA complex stabilization activity from the affinity column flowthrough. The activity coeluted from the final purification step with polypeptides of 21 and 27 kDa. The yeast gene encoding the 21-kDa protein was cloned on the basis of its N-terminal amino acid sequence. The gene, named EGD1 (enhancer of GAL4 DNA binding), encodes a highly basic protein (21% lysine and arginine) with a predicted molecular mass of 16.5 kDa. The amino acid sequence of the EGD1 product, Egd1p, is highly similar to that of the human protein BTF3 (X. M. Zheng, D. Black, P. Chambon, and J. M. Egly, Nature [London] 344:556-559, 1990). Although an egd1 null mutant was viable and Gal+, induction of the galactose-regulated genes in the egd1 mutant strain was significantly reduced when cells were shifted from glucose to galactose.

Amino Acid Sequence↗
PubMed1991

The yeast mitochondrial RNA polymerase specificity factor, MTF1, is similar to bacterial sigma factors.

We have purified the protein that confers selective promoter recognition on the core subunit of the yeast mitochondrial RNA polymerase. The N-terminal sequence of the 43-kDa specificity factor identified it as the product of the MTF1 gene described by Lisowsky and Michaelis (1988). We confirmed that MTF1 encoded the specificity factor by analyzing extracts from a yeast strain bearing a disruption of the gene. The extracts contained normal levels of core RNA polymerase but lacked selective transcription activity; adding the purified 43-kDa protein restored selective transcription. Comparison of the MTF1 protein sequence to the family of bacterial sigma factors has revealed striking similarity to domains identified with--10 promoter recognition, promoter melting, and holoenzyme stability.

Amino Acid Sequence↗
PubMed1991

The yeast IMP1 gene is allelic to GAL2.

We have found that many laboratory strains of yeast are defective in galactose metabolism owing to a recessive mutation in the previously characterized nuclear gene, IMP1. This defect leads to a requirement for mitochondrial function for growth on, and metabolism of, galactose. Genetic background affects the degree to which cells are defective. In particular, alleles of GAL3 affect the ability to score the Imp phenotype. We have found that in imp1 strains, transcriptional induction of the galactose inducible genes (GAL1, 2, 7 + 10, MEL1) is normal, but galactose transport is reduced in both rho+ and rho0 cells. This phenotype is normally associated with mutations in GAL2, the galactose permease. Although the growth phenotypes of gal2 and imp1 mutants are distinct, we found that the transformation of imp1 rho0 strains with a plasmid containing the GAL2 gene allows these strains to grow on galactose. Initial genetic analyses did not demonstrate linkage between the GAL2 and IMP1 genes owing to the effects of an unlinked gene on the Imp phenotype. By disrupting the GAL2 gene in an Imp+ background, we have shown that IMP1 and GAL2 segregate as tightly linked genes. Based on these data, we believe that imp1 is a partially defective allele of the GAL2 gene.

Alleles↗
PubMed1991

Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae.

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.

Base Sequence↗
PubMed1990

Accurate initiation by RNA polymerase II in a whole cell extract from Saccharomyces cerevisiae.

We have developed a simple procedure for isolating a transcriptional extract from whole yeast cells which obviates the requirement for nuclear isolation. Detection of accurate mRNA initiation by RNA polymerase II in the extract requires the use of a sensitive assay, recently described by Kornberg and co-workers (Lue, N. F., Flanagan, P. M., Sugimoto, K., and Kornberg, R. D. (1989) Science 246, 661-664) that involves activation by a GAL4-VP16 fusion protein and a template lacking guanosine residues in the coding strand. The extract is prepared from fresh or frozen yeast cells by disruption with glass beads and fractionation of proteins by ammonium sulfate precipitation. The alpha-amanitin-sensitive transcripts synthesized in the assay were identical to those produced in a parallel assay using a yeast nuclear extract. The activity of the whole cell extract is lower per mg of protein than a nuclear extract but proportional to the volume of the nucleus relative to the whole cell. The optimal ranges for several reaction components including template, mono- and divalent cations, and nucleotide substrate concentration were determined. Under optimal conditions the whole cell extract produced a maximum of approximately 1 X 10(-2) transcripts/template molecule in 30 min.

Amanitins↗
PubMed1990

Purification and characterization of the yeast transcriptional activator GAL4.

We have purified extensively the transcriptional activator, GAL4, from a yeast strain overexpressing the gene product from the ADH1 promoter. Our purification followed GAL4 activity by its binding to a specific DNA target sequence, using filter binding assays. No specific binding activity was detected in extracts from a strain containing a disrupted copy of the GAL4 gene. The purification protocol included fractionation of a whole cell extract by ion-exchange and DNA-affinity chromatography on a column containing a 17-base pair oligomer encoding a near consensus GAL4 binding site. Two polypeptides co-eluted with the GAL4 DNA binding activity from the DNA-affinity column. One had an apparent molecular mass of 99 kDa (the predicted size of the GAL4 protein) and cross-reacted with antibodies raised against GAL4 epitopes from fusion proteins expressed in bacterial cells. The second polypeptide did not cross-react with the anti-GAL4 antibody and is presumed to be the GAL80 transcriptional repressor based on its size (48 kDa) and known physical association with the GAL4 protein. GAL4 binding activity elutes from a gel filtration column as a 155-kDa species suggesting that it exists in solution in a heterodimer complex of one GAL4 and one GAL80 molecule. The dissociation constant of the DNA-affinity-purified GAL4-GAL80 complex for a 900-base pair DNA fragment containing the UASGAL element from the GAL1-GAL10 divergent promoter was, Kd(effective) (0.15 M KCl) = 2.4 x 10(-9) M.

Blotting, Western↗
PubMed1989

Use of yeast nuclear DNA sequences to define the mitochondrial RNA polymerase promoter in vitro.

We have extended an earlier observation that the TATA box for the nuclear GAL10 gene serves as a promoter for the mitochondrial RNA polymerase in in vitro transcription reactions (C. S. Winkley, M. J. Keller, and J. A. Jaehning, J. Biol. Chem. 260:14214-14223, 1985). In this work, we demonstrate that other nuclear genes also have upstream sequences that function in vitro as mitochondrial RNA polymerase promoters. These genes include the GAL7 and MEL1 genes, which are regulated in concert with the GAL10 gene, the sigma repetitive element, and the 2 microns plasmid origin of replication. We used in vitro transcription reactions to test a large number of nuclear DNA sequences that contain critical mitochondrial promoter sequences as defined by Biswas et al. (T. K. Biswas, J. C. Edwards, M. Rabinowitz, and G. S. Getz, J. Biol. Chem. 262:13690-13696, 1987). The results of these experiments allowed us to extend the definition of essential promoter elements. This extended sequence, -ACTATAAACGatcATAG-, was frequently found in the upstream regulatory regions of nuclear genes. On the basis of these observations, we hypothesized that either (i) a catalytic RNA polymerase related to the mitochondrial enzyme functions in the nucleus of the yeast cell or (ii) a DNA sequence recognition factor is shared by the two genetic compartments. By using cells deficient in the catalytic core of the mitochondrial RNA polymerase (rpo41-) and sensitive assays for transcripts initiating from the nuclear promoter sequences, we have conclusively ruled out a role for the catalytic RNA polymerase in synthesizing transcripts from all of the nuclear sequences analyzed. The possibility that a DNA sequence recognition factor functions in both the nucleus and the mitochondria remains to be tested.

Base Sequence↗
PubMed1988

Two forms of RPO41-dependent RNA polymerase. Regulation of the RNA polymerase by glucose repression may control yeast mitochondrial gene expression.

We have identified two chromatographically separable forms of mitochondrial RNA polymerase from Saccharomyces cerevisiae which utilize different DNA templates. One form is only active in a nonselective assay utilizing a poly[d(A-T)] template. The other form selectively initiates from a mitochondrial promoter consensus sequence. Both enzymes can be extracted from yeast mitochondria and all components are encoded by nuclear genes. The possibility that these two activities represent core and holoenzyme forms of the multicomponent mitochondrial RNA polymerase is supported by our observation that both enzymes are absent from a strain bearing a disrupted copy of the RPO41 gene (Greenleaf, A. L., Kelly, J. L., and Lehman, I. R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3391-3399). The two enzyme activities are differentially regulated by carbon source; the nonselective enzyme is repressed during growth on glucose relative to the selective enzyme. The 5-fold increase in RNA polymerase activity on a nonrepressing carbon source correlates with the increased level of transcript production from mitochondrial DNA. These results suggest that the mitochondrial RNA polymerase and, in consequence, mitochondrial transcription are regulated by carbon catabolite control.

Chromatography↗
PubMed1987

Transcription of multiple copies of the yeast GAL7 gene is limited by specific factors in addition to GAL4.

High levels of the GAL7 gene in the yeast cell appear to titrate regulatory factors and to impair transcription of related sequences. To investigate the role that the GAL regulatory factors GAL4 and GAL80 have in this process we have compared the accumulation of mRNA transcribed from single-copy (plasmid-borne GAL7 and chromosomal GAL10) and high-copy (plasmid-borne GAL7) genes in several GAL regulatory mutants. Our results show that functional GAL4 gene product is required for induction of transcription from the single- and high-copy genes. In a strain containing the GAL4 gene fused to the high expression ADH1 promoter, glucose can replace galactose to induce high levels of transcription of GAL7 and GAL10 genes, although the kinetics of accumulation induced by the two sugars are distinctly different. In the presence of high levels of GAL4, maximum accumulation of mRNA from single and high copy genes is elevated two-fold; disruption of the gal80 gene in combination with high levels of GAL4 results in a further two-fold increase in transcription. In this genetic background, galactose-induced transcription of the high copy GAL7 gene results in a greater than 50-fold increase in the levels of GAL7 mRNA, representing 30%-50% of the total cellular mRNA. Our results are consistent with a cooperative effect of saturation of multiple GAL4 DNA binding sites and with a limiting factor, in addition to GAL4, that is required for transcription of the GAL genes.

Alcohol Dehydrogenase↗
PubMed1986

mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.

Amanitins↗
PubMed1985

A transcription map of a yeast centromere plasmid: unexpected transcripts and altered gene expression.

YCp19 is a yeast centromere plasmid capable of autonomous replication in both yeast and E. coli (J. Mol. Biol., 158: 157-179, 1982). It is stably maintained as a single copy in the yeast cell and is therefore a model yeast "minichromosome" and cloning vector. We have located the positions and measured the abundance of the in vivo yeast transcripts from YCp19. Transcripts from the selectable marker genes TRP1 and URA3 were present at increased levels relative to chromosomal copies of the genes. Unanticipated transcripts from the yeast CEN4 and E. coli pBR322 sequences were also found. Although much of the plasmid vector is actively transcribed in vivo, the regions around the most useful cloning sites (BamHI, EcoRI, SalI) are free of transcripts. We have analyzed transcription of BamHI inserts containing promoter variants of the HIS3 gene and determined that although initiation events are accurate, plasmid context may alter levels of gene expression.

Centromere↗
PubMed1985

A multicomponent mitochondrial RNA polymerase from Saccharomyces cerevisiae.

Using a whole cell extract from Saccharomyces cerevisiae (bakers' yeast) we have been able to detect a selective RNA polymerase activity originally identified in purified yeast mitochondria (Levens, D., Morimoto, R., and Rabinowitz, M. (1981) J. Biol. Chem. 256, 1466-1473). We have shown that in in vitro transcription reactions this activity recognizes a consensus mitochondrial promoter sequence ATA-TAAGTA (Osinga, K. A., DeHaan, M., Christianson, T., and Tabak, H. F. (1982) Nucleic Acids Res. 10, 7993-8006) in the upstream region of the nuclear GAL10 gene as well as promoters from yeast mitochondrial DNA. Using these promoter-containing templates for in vitro assays, we have chromatographically separated the mitochondrial specific RNA polymerase activity from the three nuclear RNA polymerases (I, II, and III). Further characterization has revealed that this preparation has distinctive properties on two different types of DNA templates, poly[d(AT)] and cloned DNA containing mitochondrial promoters. Salt and divalent cation optima and substrate saturation kinetics are different for the two types of templates. Using promoter-containing DNA as an assay template, we have chromatographically dissociated the RNA polymerase activity into two nonfunctional components. Selective transcription of the GAL10 template is restored when the two components are recombined. It is possible that the RNA polymerase active on poly[d(AT)] is a nonspecific component of the selective transcription apparatus or that two distinct RNA polymerases are present in the preparation.

Base Sequence↗