SPT15, the gene encoding the yeast TATA binding factor TFIID, is required for normal transcription initiation in vivo

Cell ◽  
1989 ◽  
Vol 58 (6) ◽  
pp. 1183-1191 ◽  
Author(s):  
David M. Eisenmann ◽  
Catherine Dollard ◽  
Fred Winston
Keyword(s):  
Transcription Initiation ◽  
Gene Encoding ◽  
Binding Factor

scholarly journals The conserved carboxy-terminal domain of Saccharomyces cerevisiae TFIID is sufficient to support normal cell growth

1991 ◽  
Vol 11 (10) ◽  
pp. 4809-4821
Author(s):  
D Poon ◽  
S Schroeder ◽  
C K Wang ◽  
T Yamamoto ◽  
M Horikoshi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Growth ◽  
Transcription Initiation ◽  
Mutant Form ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Gene Encoding ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal

We have examined the structure-function relationships of TFIID through in vivo complementation tests. A yeast strain was constructed which lacked the chromosomal copy of SPT15, the gene encoding TFIID, and was therefore dependent on a functional plasmid-borne wild-type copy of this gene for viability. By using the plasmid shuffle technique, the plasmid-borne wild-type TFIID gene was replaced with a family of plasmids containing a series of systematically mutated TFIID genes. These various forms of TFIID were expressed from three different promoter contexts of different strengths, and the ability of each mutant form of TFIID to complement our chromosomal TFIID null allele was assessed. We found that the first 61 amino acid residues of TFIID are totally dispensable for vegetative cell growth, since yeast strains containing this deleted form of TFIID grow at wild-type rates. Amino-terminally deleted TFIID was further shown to be able to function normally in vivo by virtue of its ability both to promote accurate transcription initiation from a large number of different genes and to interact efficiently with the Gal4 protein to activate transcription of GAL1 with essentially wild-type kinetics. Any deletion removing sequences from within the conserved carboxy-terminal region of S. cerevisiae TFIID was lethal. Further, the exact sequence of the conserved carboxy-terminal portion of the molecule is critical for function, since of several heterologous TFIID homologs tested, only the highly related Schizosaccharomyces pombe gene could complement our S. cerevisiae TFIID null mutant. Taken together, these data indicate that all important functional domains of TFIID appear to lie in its carboxy-terminal 179 amino acid residues. The significance of these findings regarding TFIID function are discussed.

scholarly journals The conserved carboxy-terminal domain of Saccharomyces cerevisiae TFIID is sufficient to support normal cell growth.

1991 ◽  
Vol 11 (10) ◽  
pp. 4809-4821 ◽  
Author(s):  
D Poon ◽  
S Schroeder ◽  
C K Wang ◽  
T Yamamoto ◽  
M Horikoshi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Growth ◽  
Transcription Initiation ◽  
Mutant Form ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Gene Encoding ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal

We have examined the structure-function relationships of TFIID through in vivo complementation tests. A yeast strain was constructed which lacked the chromosomal copy of SPT15, the gene encoding TFIID, and was therefore dependent on a functional plasmid-borne wild-type copy of this gene for viability. By using the plasmid shuffle technique, the plasmid-borne wild-type TFIID gene was replaced with a family of plasmids containing a series of systematically mutated TFIID genes. These various forms of TFIID were expressed from three different promoter contexts of different strengths, and the ability of each mutant form of TFIID to complement our chromosomal TFIID null allele was assessed. We found that the first 61 amino acid residues of TFIID are totally dispensable for vegetative cell growth, since yeast strains containing this deleted form of TFIID grow at wild-type rates. Amino-terminally deleted TFIID was further shown to be able to function normally in vivo by virtue of its ability both to promote accurate transcription initiation from a large number of different genes and to interact efficiently with the Gal4 protein to activate transcription of GAL1 with essentially wild-type kinetics. Any deletion removing sequences from within the conserved carboxy-terminal region of S. cerevisiae TFIID was lethal. Further, the exact sequence of the conserved carboxy-terminal portion of the molecule is critical for function, since of several heterologous TFIID homologs tested, only the highly related Schizosaccharomyces pombe gene could complement our S. cerevisiae TFIID null mutant. Taken together, these data indicate that all important functional domains of TFIID appear to lie in its carboxy-terminal 179 amino acid residues. The significance of these findings regarding TFIID function are discussed.

scholarly journals Functional Interaction between Ssu72 and the Rpb2 Subunit of RNA Polymerase II in Saccharomyces cerevisiae

2000 ◽  
Vol 20 (22) ◽  
pp. 8343-8351 ◽  
Author(s):  
Donald L. Pappas ◽  
Michael Hampsey
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Essential Gene ◽  
Functional Interaction ◽  
Gene Encoding ◽  
General Transcription Factor ◽  
Physical Interactions ◽  
Rnap Ii

ABSTRACT SSU72 is an essential gene encoding a phylogenetically conserved protein of unknown function that interacts with the general transcription factor TFIIB. A recessive ssu72-1 allele was identified as a synthetic enhancer of a TFIIB (sua7-1) defect, resulting in a heat-sensitive (Ts−) phenotype and a dramatic downstream shift in transcription start site selection. Here we describe a new allele, ssu72-2, that confers a Ts− phenotype in a SUA7 wild-type background. In an effort to further define Ssu72, we isolated suppressors of thessu72-2 mutation. One suppressor is allelic toRPB2, the gene encoding the second-largest subunit of RNA polymerase II (RNAP II). Sequence analysis of the rpb2-100suppressor defined a cysteine replacement of the phylogenetically invariant arginine residue at position 512 (R512C), located within homology block D of Rpb2. The ssu72-2 andrpb2-100 mutations adversely affected noninduced gene expression, with no apparent effects on activated transcription in vivo. Although isolated as a suppressor of the ssu72-2Ts− defect, rpb2-100 enhanced the transcriptional defects associated with ssu72-2. The Ssu72 protein interacts directly with purified RNAP II in a coimmunoprecipitation assay, suggesting that the genetic interactions between ssu72-2 and rpb2-100 are a consequence of physical interactions. These results define Ssu72 as a highly conserved factor that physically and functionally interacts with the RNAP II core machinery during transcription initiation.

Sp1, a CAAT-binding factor, and the adenovirus major late promoter transcription factor interact with functional regions of the gamma-fibrinogen promoter

1988 ◽  
Vol 8 (6) ◽  
pp. 2628-2637
Author(s):  
J G Morgan ◽  
G Courtois ◽  
G Fourel ◽  
L A Chodosh ◽  
L Campbell ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Internal Deletion ◽  
Developmentally Regulated ◽  
Mrna Accumulation ◽  
Deletion Mutations ◽  
Functional Regions ◽  
Binding Factor

To study the factors which influence the coordinately and developmentally regulated expression of the three adjacent fibrinogen genes, we have defined the functional regions of the gamma-fibrinogen promoter and the proteins which bind to them. Using a series of 5' and internal deletion mutations, we found that sequences between 88 and 43 base pairs (bp) upstream of the gamma-fibrinogen transcription initiation site functioned in cis to direct properly initiated mRNA accumulation in transfected hepatocytes. The efficient function of these sequences was highly distance dependent, since transcriptional activity decreased by 92% when they were moved 32 bp upstream of the TATA box. We demonstrated that two known and one putative transcriptional factors interacted with this 47-bp sequence. The transcription factor Sp1 interacted with sequences between -51 and -46 as demonstrated by protection from DNase I digestion with the purified protein. Directly adjacent to the Sp1 site, between nucleotides -66 and -53, there was a sequence which bound a CAAT-binding factor. Finally, sequences just 5' to the CAAT factor-binding site interacted with the adenovirus major late transcriptional factor as previously demonstrated. Internal deletion mutations which disrupt these interactions diminished the activity of the promoter in vivo. One consequence of the interaction of these proteins is that a bend is placed in the DNA at or near their sites of interaction.

scholarly journals Sp1, a CAAT-binding factor, and the adenovirus major late promoter transcription factor interact with functional regions of the gamma-fibrinogen promoter.

1988 ◽  
Vol 8 (6) ◽  
pp. 2628-2637 ◽  
Author(s):  
J G Morgan ◽  
G Courtois ◽  
G Fourel ◽  
L A Chodosh ◽  
L Campbell ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Internal Deletion ◽  
Developmentally Regulated ◽  
Mrna Accumulation ◽  
Deletion Mutations ◽  
Functional Regions ◽  
Binding Factor

To study the factors which influence the coordinately and developmentally regulated expression of the three adjacent fibrinogen genes, we have defined the functional regions of the gamma-fibrinogen promoter and the proteins which bind to them. Using a series of 5' and internal deletion mutations, we found that sequences between 88 and 43 base pairs (bp) upstream of the gamma-fibrinogen transcription initiation site functioned in cis to direct properly initiated mRNA accumulation in transfected hepatocytes. The efficient function of these sequences was highly distance dependent, since transcriptional activity decreased by 92% when they were moved 32 bp upstream of the TATA box. We demonstrated that two known and one putative transcriptional factors interacted with this 47-bp sequence. The transcription factor Sp1 interacted with sequences between -51 and -46 as demonstrated by protection from DNase I digestion with the purified protein. Directly adjacent to the Sp1 site, between nucleotides -66 and -53, there was a sequence which bound a CAAT-binding factor. Finally, sequences just 5' to the CAAT factor-binding site interacted with the adenovirus major late transcriptional factor as previously demonstrated. Internal deletion mutations which disrupt these interactions diminished the activity of the promoter in vivo. One consequence of the interaction of these proteins is that a bend is placed in the DNA at or near their sites of interaction.

scholarly journals The Multifaceted Roles of MicroRNAs in Cystic Fibrosis

Diagnostics ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1102
Author(s):  
Fatima Domenica Elisa De Palma ◽  
Valeria Raia ◽  
Guido Kroemer ◽  
Maria Chiara Maiuri
Keyword(s):  
Cystic Fibrosis ◽  
Respiratory Infections ◽  
Current Knowledge ◽  
Pancreatic Insufficiency ◽  
Clinical Manifestations ◽  
Predictive Biomarkers ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Multisystemic Disease

Cystic fibrosis (CF) is a lifelong disorder affecting 1 in 3500 live births worldwide. It is a monogenetic autosomal recessive disease caused by loss-of-function mutations in the gene encoding the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR), the impairment of which leads to ionic disequilibria in exocrine organs. This translates into a chronic multisystemic disease characterized by airway obstruction, respiratory infections, and pancreatic insufficiency as well as hepatobiliary and gastrointestinal dysfunction. Molecular characterization of the mutational heterogeneity of CFTR (affected by more than 2000 variants) improved the understanding and management of CF. However, these CFTR variants are linked to different clinical manifestations and phenotypes, and they affect response to treatments. Expanding evidence suggests that multisystemic disease affects CF pathology via impairing either CFTR or proteins regulated by CFTR. Thus, altering the expression of miRNAs in vivo could constitute an appealing strategy for developing new CF therapies. In this review, we will first describe the pathophysiology and clinical management of CF. Then, we will summarize the current knowledge on altered miRNAs in CF patients, with a focus on the miRNAs involved in the deregulation of CFTR and in the modulation of inflammation. We will highlight recent findings on the potential utility of measuring circulating miRNAs in CF as diagnostic, prognostic, and predictive biomarkers. Finally, we will provide an overview on potential miRNA-based therapeutic approaches.

scholarly journals A 5′ Splice Site Enhances the Recruitment of Basal Transcription Initiation Factors In Vivo

2008 ◽  
Vol 29 (2) ◽  
pp. 271-278 ◽  
Author(s):  
Christian Kroun Damgaard ◽  
Søren Kahns ◽  
Søren Lykke-Andersen ◽  
Anders Lade Nielsen ◽  
Torben Heick Jensen ◽  
...  
Keyword(s):  
Splice Site ◽  
Transcription Initiation ◽  
Basal Transcription ◽  
Initiation Factors

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

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