Functional domains of a positive regulatory protein, PHO4, for transcriptional control of the phosphatase regulon in Saccharomyces cerevisiae.

The PHO4 gene encodes a positive regulatory factor involved in regulating transcription of various genes in the phosphatase regulon of Saccharomyces cerevisiae. Besides its own coding region, the 1.8-kilobase PHO4 transcript contains a coding region for a mitochondrial protein which does not appear to be translated. Four functional domains were found in the PHO4 protein, which consists of 312 amino acid (aa) residues as deduced from the open reading frame of PHO4. A gel retardation assay with beta-galactosidase::PHO4 fused protein revealed that the 85-aa C terminus is the domain responsible for binding to the promoter DNA of PHO5, a gene under the control of PHO4. This region has similarities with the amphipathic helix-loop-helix motif of c-myc protein. Determination of the nucleotide sequences of four PHO4c mutant alleles and insertion and deletion analyses of PHO4 DNA indicated that a region from aa 163 to 202 is involved in interaction with a negative regulatory factor PHO80. Complementation of a pho4 null allele with the modified PHO4 DNAs suggested that the N-terminal region (1 to 109 aa), which is rich in acidic aa, is the transcriptional activation domain. The deleterious effects of various PHO4 mutations on the constitutive transcription of PHO5 in PHO4c mutant cells suggested that the region from aa 203 to 227 is involved in oligomerization of the PHO4 protein.

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Functional domains of a negative regulatory protein, GAL80, of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.3009 ◽

1989 ◽

Vol 9 (7) ◽

pp. 3009-3017 ◽

Cited By ~ 22

Author(s):

Y Nogi ◽

T Fukasawa

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Regulatory Protein ◽

Subcellular Fractionation ◽

Functional Domains ◽

Amino Acid Residues ◽

Coding Region ◽

Interaction Domain ◽

Repression Domain

To study the functional domains of a transcriptional repressor encoded by the GAL80 gene of Saccharomyces cerevisiae, we constructed various deletion and insertion mutations in the GAL80 coding region and determined the ability of these mutations to repress synthesis of galactose-metabolizing enzymes as well as the capacity of the mutant proteins to respond to the inducer. Two regions, from amino acids 1 to 321 and from amino acids 341 to 423, in the total sequence of 435 amino acids were required for repression. The internal region from amino acids 321 to 340 played a role in the response to the inducer. The 12 amino acids at the carboxy terminus were dispensable for normal functioning of the GAL80 protein. Using indirect immunofluorescence and subcellular fractionation techniques, we also found that two distinct regions (amino acids 1 to 109 and 342 to 405) within the putative repression domain were capable of directing cytoplasmically synthesized Escherichia coli beta-galactosidase to the yeast nucleus. In addition, three gal80 mutations were mapped at amino acid residues 183, 298, and 310 in the domain required for repression. On the basis of these results, we suggest that the GAL80 protein consists of a repression domain located in two separate regions (amino acid residues 1 to 321 and 341 to 423) that are interrupted by an inducer interaction domain (residues 322 to 340) and two nuclear localization domains (1 to 109 and 342 to 405) that overlap the repression domains.

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Functional domains of a negative regulatory protein, GAL80, of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.3009-3017.1989 ◽

1989 ◽

Vol 9 (7) ◽

pp. 3009-3017

Author(s):

Y Nogi ◽

T Fukasawa

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Regulatory Protein ◽

Subcellular Fractionation ◽

Functional Domains ◽

Amino Acid Residues ◽

Coding Region ◽

Interaction Domain ◽

Repression Domain

To study the functional domains of a transcriptional repressor encoded by the GAL80 gene of Saccharomyces cerevisiae, we constructed various deletion and insertion mutations in the GAL80 coding region and determined the ability of these mutations to repress synthesis of galactose-metabolizing enzymes as well as the capacity of the mutant proteins to respond to the inducer. Two regions, from amino acids 1 to 321 and from amino acids 341 to 423, in the total sequence of 435 amino acids were required for repression. The internal region from amino acids 321 to 340 played a role in the response to the inducer. The 12 amino acids at the carboxy terminus were dispensable for normal functioning of the GAL80 protein. Using indirect immunofluorescence and subcellular fractionation techniques, we also found that two distinct regions (amino acids 1 to 109 and 342 to 405) within the putative repression domain were capable of directing cytoplasmically synthesized Escherichia coli beta-galactosidase to the yeast nucleus. In addition, three gal80 mutations were mapped at amino acid residues 183, 298, and 310 in the domain required for repression. On the basis of these results, we suggest that the GAL80 protein consists of a repression domain located in two separate regions (amino acid residues 1 to 321 and 341 to 423) that are interrupted by an inducer interaction domain (residues 322 to 340) and two nuclear localization domains (1 to 109 and 342 to 405) that overlap the repression domains.

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The GIS2 Gene Is Repressed by a Zinc-Regulated Bicistronic RNA in Saccharomyces cerevisiae

Genes ◽

10.3390/genes9090462 ◽

2018 ◽

Vol 9 (9) ◽

pp. 462 ◽

Cited By ~ 2

Author(s):

Janet Taggart ◽

Yirong Wang ◽

Erin Weisenhorn ◽

Colin W. MacDiarmid ◽

Jason Russell ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase Ii ◽

Zinc Deficiency ◽

Transcriptional Activation ◽

Open Reading Frames ◽

Zinc Homeostasis ◽

Protein Accumulation ◽

Coding Region ◽

Yeast Saccharomyces Cerevisiae ◽

Short Transcript

Zinc homeostasis is essential for all organisms. The Zap1 transcriptional activator regulates these processes in the yeast Saccharomyces cerevisiae. During zinc deficiency, Zap1 increases expression of zinc transporters and proteins involved in adapting to the stress of zinc deficiency. Transcriptional activation by Zap1 can also repress expression of some genes, e.g., RTC4. In zinc-replete cells, RTC4 mRNA is produced with a short transcript leader that is efficiently translated. During deficiency, Zap1-dependent expression of an RNA with a longer transcript leader represses the RTC4 promoter. This long leader transcript (LLT) is not translated due to the presence of small open reading frames upstream of the RTC4 coding region. In this study, we show that the RTC4 LLT RNA also plays a second function, i.e., repression of the adjacent GIS2 gene. In generating the LLT transcript, RNA polymerase II transcribes RTC4 through the GIS2 promoter. Production of the LLT RNA correlates with the decreased expression of GIS2 mRNA and mutations that prevent synthesis of the LLT RNA or terminate it before the GIS2 promoter renders GIS2 mRNA expression and Gis2 protein accumulation constitutive. Thus, we have discovered an unusual regulatory mechanism that uses a bicistronic RNA to control two genes simultaneously.

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Functional domains of interferon regulatory factor I (IRF-1)

Biochemical Journal ◽

10.1042/bj3350147 ◽

1998 ◽

Vol 335 (1) ◽

pp. 147-157 ◽

Cited By ~ 67

Author(s):

Fred SCHAPER ◽

Sabine KIRCHHOFF ◽

Guido POSERN ◽

Mario KÖSTER ◽

André OUMARD ◽

...

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Nuclear Translocation ◽

Consensus Sequence ◽

Binding Domain ◽

Functional Domains ◽

Dna Binding Domain ◽

Transcriptional Activation Domain

Interferon (IFN) regulatory factors (IRFs) are a family of transcription factors among which are IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). These factors share sequence homology in the N-terminal DNA-binding domain. IRF-1 and IRF-2 are further related and have additional homologous sequences within their C-termini. Whereas IRF-2 and ICSBP are identified as transcriptional repressors, IRF-1 is an activator. In the present work, the identification of functional domains in murine IRF-1 with regard to DNA-binding, nuclear translocation, heterodimerization with ICSBP and transcriptional activation are demonstrated. The minimal DNA-binding domain requires the N-terminal 124 amino acids plus an arbitrary C-terminal extension. By using mutants of IRF-1 fusion proteins with green fluorescent protein and monitoring their distribution in living cells, a nuclear location signal (NLS) was identified and found to be sufficient for nuclear translocation. Heterodimerization was confirmed by a two-hybrid system adapted to mammalian cells. The heterodimerization domain in IRF-1 was defined by studies in vitroand was shown to be homologous with a sequence in IRF-2, suggesting that IRF-2 also heterodimerizes with ICSBP through this sequence. An acidic domain in IRF-1 was found to be required and to be sufficient for transactivation. Epitope mapping of IRF-1 showed that regions within the NLS, the heterodimerization domain and the transcriptional activation domain are exposed for possible contacts with interacting proteins.

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Differential transcriptional activation by v-myb and c-myb in animal cells and Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4423 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4423-4431 ◽

Cited By ~ 12

Author(s):

R H Chen ◽

J S Lipsick

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Transcriptional Activation ◽

Animal Cell ◽

Cell Protein ◽

Detectable Effect ◽

Animal Cells ◽

Transcriptional Activation Domain ◽

Recognition Element ◽

Myb Proteins

The v-myb oncogene and its cellular homolog c-myb encode sequence-specific DNA-binding proteins which regulate transcription from promoters containing Myb-binding sites in animal cells. We have developed a Saccharomyces cerevisiae system to assay transcriptional activation by v-Myb and c-Myb. In yeast strains containing integrated reporter genes, activation was strictly dependent upon both the Myb DNA-binding domain and the Myb recognition element. BAS1, an endogenous Myb-related yeast protein, was not required for transactivation by animal Myb proteins and by itself had no detectable effect on a Myb reporter gene. Deletion analyses demonstrated that a domain of v-Myb C terminal to the previously mapped Myb transcriptional activation domain was required for transactivation in animal cells but not in S. cerevisiae. The same domain is also required for the efficient transformation of myeloid cells by v-Myb. In contrast to results in animal cells, in S. cerevisiae the full-length c-Myb was a much stronger transactivator than a protein bearing the oncogenic N- and C-terminal truncations of v-Myb. These results imply that negative regulation of c-Myb by its own termini requires an additional animal cell protein or small molecule that is not present in S. cerevisiae.

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A Region of the Transmembrane Regulatory Protein ToxR That Tethers the Transcriptional Activation Domain to the Cytoplasmic Membrane Displays Wide Divergence amongVibrio Species

Journal of Bacteriology ◽

10.1128/jb.182.2.526-528.2000 ◽

2000 ◽

Vol 182 (2) ◽

pp. 526-528 ◽

Cited By ~ 59

Author(s):

Carlos R. Osorio ◽

Karl E. Klose

Keyword(s):

Transcriptional Activation ◽

Cytoplasmic Membrane ◽

Transmembrane Domain ◽

Regulatory Protein ◽

Amino Acid Sequences ◽

Activation Domain ◽

Ancestral Gene ◽

Transcriptional Activation Domain ◽

Cytoplasmic Dna ◽

The Family

ABSTRACT The virulence regulatory protein ToxR of Vibrio cholerae is unique in that it contains a cytoplasmic DNA-binding–transcriptional activation domain, a transmembrane domain, and a periplasmic domain. Although ToxR and other transmembrane transcriptional activators have been discovered in other bacteria, little is known about their mechanism of activation. Utilizing degenerate oligonucleotides and PCR, we have amplified internaltoxR gene sequences from seven Vibrio andPhotobacterium species and subspecies, demonstrating thattoxR is an ancestral gene of the familyVibrionaceae. Sequence alignment of all available ToxR amino acid sequences revealed a region between the transcriptional activation and transmembrane domains that displays wide divergence among Vibrio species. We hypothesize that this region merely tethers the transcriptional activation domain to the cytoplasmic membrane and thus can tolerate wide divergence and multiple insertions and deletions. The divergence in the tether region at the nucleotide level may provide a useful tool for the distinction ofVibrio and Photobacterium species.

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Plasmodium Falciparum Regulates Transcriptional Output by Modulating The Activity of RNA Polymerase II Rather Than Its Recruitment

10.21203/rs.3.rs-74869/v1 ◽

2020 ◽

Author(s):

Ragini Rai Khandelia ◽

Aarthi Mohan ◽

Lei Zhu ◽

Jie Zheng ◽

Mark Stephen Featherstone ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Transcriptional Control ◽

Wide Distribution ◽

Developmental Cycle ◽

Mrna Levels ◽

Strong Positive Correlation ◽

Coding Region

Abstract Background The asexual phase of the unicellular eukaryotic parasite Plasmodium falciparum takes place in the human red blood cell (RBC). During the 48 h between RBC infection and the release of new progeny – the intraerythrocytic developmental cycle (IDC) – transcripts levels for most of the parasite’s ~5,772 genes are set by the integration of transcriptional and post-transcriptional processes. Transcription of eukaryotic protein coding genes is carried out by RNA polymerase II (RNAPII) whose functional states are reflected by the phosphorylation status of serine residues at positions 2 and 5 within the so-called heptad repeats within the C-terminal domain (CTD) of the catalytic subunit, RPB1; unphosphorylated or Ser5-phosphorylated RNAPII correspond to uninitiated and initiating states, while phosphorylation at Ser2 is associated with the processive elongating form of the polymerase. Transcriptional control over the IDC could impinge on RNAPII recruitment, initiation, and conversion to processive elongation. To distinguish the contribution of these potential regulatory steps to transcriptional control during infection, we used ChIP-seq and RNA-seq to determine the genome-wide distribution of three RNAPII phosphoisoforms as well as total RNAPII and correlated occupancy with mRNA levels across the three stages of the IDC.ResultsWe find that most genes are occupied by RNAPII along the length of the coding region at all stages of the IDC, regardless of transcript output, with RNAPII accumulation of all forms strongly biased toward the coding region. Total RNAPII levels at a given gene are relatively constant across the IDC. By contrast, occupancy by the elongating form of the polymerase shows a strong positive correlation with mRNA abundance. ConclusionsThese observations reveal that transcriptional activation during the IDC is not regulated primarily at the level of RNAPII recruitment, but rather through the conversion of RNAPII to its processive elongating form. Our results have implications for the expected roles of enhancers and transcription factors in the parasite and support a major role for the control of transcriptional pausing.

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