Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro.

Fusion proteins known to activate transcription in vivo were tested for the ability to stimulate transcription in vitro in a recently developed Saccharomyces cerevisiae RNA polymerase II transcription system. One fusion protein, whose activation domain was derived from the herpesvirus transcriptional activator VP16, gave more than 100-fold stimulation in the in vitro system. The order of effects of the various proteins was the same for transcription in vitro and in vivo, suggesting that the natural mechanism of activation is preserved in vitro.

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Heterogeneous nuclear ribonucleoprotein K is a transcription factor.

Molecular and Cellular Biology ◽

10.1128/mcb.16.5.2350 ◽

1996 ◽

Vol 16 (5) ◽

pp. 2350-2360 ◽

Cited By ~ 243

Author(s):

E F Michelotti ◽

G A Michelotti ◽

A I Aronsohn ◽

D Levens

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Hnrnp K ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Transcription In Vitro ◽

Nuclear Ribonucleoprotein ◽

Rna Polymerase Ii Transcription

The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Spl and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind. The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro. The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence. Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing single-stranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted. Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA-binding protein (TBP)-TBP-associated factor complex. Coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K. Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor.

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Evidence for the Involvement of the Glc7-Reg1 Phosphatase and the Snf1-Snf4 Kinase in the Regulation of INO1 Transcription in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/152.1.73 ◽

1999 ◽

Vol 152 (1) ◽

pp. 73-87 ◽

Cited By ~ 6

Author(s):

Margaret K Shirra ◽

Karen M Arndt

Keyword(s):

Rna Polymerase Ii ◽

Glucose Repression ◽

Snf1 Kinase ◽

Glucose Levels ◽

Recessive Mutations ◽

Mutant Strains ◽

Rna Polymerase Ii Transcription ◽

Two Hybrid

AbstractBinding of the TATA-binding protein (TBP) to the promoter is a pivotal step in RNA polymerase II transcription. To identify factors that regulate TBP, we selected for suppressors of a TBP mutant that exhibits promoter-specific defects in activated transcription in vivo and severely reduced affinity for TATA boxes in vitro. Dominant mutations in SNF4 and recessive mutations in REG1, OPI1, and RTF2 were isolated that specifically suppress the inositol auxotrophy of the TBP mutant strains. OPI1 encodes a repressor of INO1 transcription. REG1 and SNF4 encode regulators of the Glc7 phosphatase and Snf1 kinase, respectively, and have well-studied roles in glucose repression. In two-hybrid assays, one SNF4 mutation enhances the interaction between Snf4 and Snf1. Suppression of the TBP mutant by our reg1 and SNF4 mutations appears unrelated to glucose repression, since these mutations do not alleviate repression of SUC2, and glucose levels have little effect on INO1 transcription. Moreover, mutations in TUP1, SSN6, and GLC7, but not HXK2 and MIG1, can cause suppression. Our data suggest that association of TBP with the TATA box may be regulated, directly or indirectly, by a substrate of Snf1. Analysis of INO1 transcription in various mutant strains suggests that this substrate is distinct from Opi1.

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Accurate transcription of a plant mitochondrial gene in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2035-2039.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2035-2039

Author(s):

P J Hanic-Joyce ◽

M W Gray

Keyword(s):

Mitochondrial Gene ◽

Plant Mitochondria ◽

In Vitro Transcription ◽

Dna Templates ◽

In Vitro System ◽

Transcription System ◽

Translation Initiation Codon ◽

Mitochondrial Extract

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

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Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system

Biochemical Journal ◽

10.1042/bj3280669 ◽

1997 ◽

Vol 328 (2) ◽

pp. 669-675 ◽

Cited By ~ 17

Author(s):

L. Tamara DOERING ◽

Randy SCHEKMAN

Keyword(s):

Saccharomyces Cerevisiae ◽

Fusion Proteins ◽

Attachment Site ◽

Gpi Anchor ◽

Mating Pheromone ◽

Peptide Substrates ◽

In Vitro System ◽

Glycosyl Phosphatidylinositol

The yeast mating pheromone precursor prepro-alpha factor was fused to C-terminal signals for glycosyl-phosphatidylinositol (GPI) anchor attachment, based on the sequence of the Saccharomyces cerevisiae protein Gas1p. Maturation of fusion proteins expressed in vivo required the presence of both a functional GPI attachment site and the synthesis of GPI precursors. Constructs were translated in vitro for use in cell-free studies of glycolipid attachment. The radiolabelled polypeptides were post-translationally translocated into yeast microsomes, where at least one third of the molecules received a GPI anchor. This approach offers distinct advantages over anchor attachment reactions that require co-translational translocation of secretory peptide substrates.

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[38] RNA polymerase II transcription in vitro

Guide to Yeast Genetics and Molecular Biology - Methods in Enzymology ◽

10.1016/0076-6879(91)94041-a ◽

1991 ◽

pp. 545-550 ◽

Cited By ~ 15

Author(s):

Neal F. Lue ◽

Peter M. Flanagan ◽

Raymond J. Kelleher ◽

Aled M. Edwards ◽

Roger D. Kornberg

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription In Vitro ◽

Rna Polymerase Ii Transcription

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Accurate transcription of a plant mitochondrial gene in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2035 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2035-2039 ◽

Cited By ~ 37

Author(s):

P J Hanic-Joyce ◽

M W Gray

Keyword(s):

Mitochondrial Gene ◽

Plant Mitochondria ◽

In Vitro Transcription ◽

Dna Templates ◽

In Vitro System ◽

Transcription System ◽

Translation Initiation Codon ◽

Mitochondrial Extract

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

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In vitro initiation of transcription by RNA polymerase II on in vivo-assembled chromatin templates.

Molecular and Cellular Biology ◽

10.1128/mcb.12.4.1639 ◽

1992 ◽

Vol 12 (4) ◽

pp. 1639-1651 ◽

Cited By ~ 2

Author(s):

S C Batson ◽

R Sundseth ◽

C V Heath ◽

M Samuels ◽

U Hansen

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Viral Dna ◽

Dna Templates ◽

Initiation Of Transcription ◽

Transcription In Vitro ◽

Alpha Amanitin

We have studied the initiation of transcription in vitro by RNA polymerase II on simian virus 40 (SV40) minichromosomal templates isolated from infected cells. The efficiency and pattern of transcription from the chromatin templates were compared with those from viral DNA templates by using two in vitro transcription systems, either HeLa whole-cell extract or basal transcription factors, RNA polymerase II, and one of two SV40 promoter-binding transcription factors, LSF and Sp1. Dramatic increases in numbers of transcripts upon addition of transcription extract and different patterns of usage of the multiple SV40 initiation sites upon addition of Sp1 versus LSF strongly suggested that transcripts were being initiated from the minichromosomal templates in vitro. That the majority of transcripts from the minichromosomes were due to initiation de novo was demonstrated by the efficient transcription observed in the presence of alpha-amanitin, which inhibited minichromosome-associated RNA polymerase II, and an alpha-amanitin-resistant RNA polymerase II, which initiated transcription in vitro. The pattern of transcription from the SV40 late and early promoters on the minichromosomal templates was similar to the in vivo pattern of transcription during the late stages of viral infection and was distinct from the pattern of transcription generated from viral DNA in vitro. In particular, the late promoter of the minichromosomal templates was transcribed with high efficiency, similar to viral DNA templates, while the early-early promoter of the minichromosomal templates was inhibited 10- to 15-fold. Finally, the number of minichromosomes competent to initiate transcription in vitro exceeded the amount actively being transcribed in vivo.

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