RESUMO
Spt5 is an essential and conserved factor that functions in transcription and co-transcriptional processes. However, many aspects of the requirement for Spt5 in transcription are poorly understood. We have analyzed the consequences of Spt5 depletion in Schizosaccharomyces pombe using four genome-wide approaches. Our results demonstrate that Spt5 is crucial for a normal rate of RNA synthesis and distribution of RNAPII over transcription units. In the absence of Spt5, RNAPII localization changes dramatically, with reduced levels and a relative accumulation over the first â¼500 bp, suggesting that Spt5 is required for transcription past a barrier. Spt5 depletion also results in widespread antisense transcription initiating within this barrier region. Deletions of this region alter the distribution of RNAPII on the sense strand, suggesting that the barrier observed after Spt5 depletion is normally a site at which Spt5 stimulates elongation. Our results reveal a global requirement for Spt5 in transcription elongation.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , RNA Antissenso/biossíntese , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Elongação da Transcrição Genética , Fatores de Elongação da Transcrição/metabolismo , Proteínas Cromossômicas não Histona/genética , Biologia Computacional , Bases de Dados Genéticas , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Genótipo , Mutação , Fenótipo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Splicing de RNA , RNA Antissenso/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Tempo , Fatores de Elongação da Transcrição/genéticaRESUMO
The Saccharomyces cerevisiae INO1 gene encodes the structural enzyme inositol-3-phosphate synthase for the synthesis de novo of inositol and inositol-containing phospholipids. The transcription of INO1 is completely derepressed in the absence of inositol and choline (I(-) C(-)). Derepression requires the binding of the Ino2p-Ino4p basic helix-loop-helix (bHLH) heterodimer to the UAS(INO) promoter element. We report here the requirement of a third bHLH protein, centromere-binding factor 1 (Cbf1p), for the complete derepression of INO1 transcription. We found that Cbf1p regulates INO1 transcription by binding to sites distal to the INO1 promoter and encompassing the upstream SNA3 open reading frame (ORF) and promoter. The binding of Cbf1p requires Ino2p-Ino4p binding to the UAS(INO) sites in the INO1 promoter and vice versa, suggesting a cooperative mechanism. Furthermore, Cbf1p binding to the upstream sites was required for the binding of the ISW2 chromatin-remodeling complex to the Ino2p-Ino4p-binding sites on the INO1 promoter. Consistent with this, ISW2 was also required for the complete derepression of INO1 transcription.
Assuntos
Adenosina Trifosfatases/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Montagem e Desmontagem da Cromatina , Regulação Fúngica da Expressão Gênica , Mio-Inositol-1-Fosfato Sintase/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cromatina/genética , Cromatina/metabolismo , Dimerização , Mio-Inositol-1-Fosfato Sintase/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The auxin-inducible degron (AID) is a powerful tool that is used for depletion of proteins to study their function in vivo. This method can conditionally induce the degradation of any protein by the proteasome simply by the addition of the plant hormone auxin. This approach is particularly valuable to study the function of essential proteins. The protocols provided here describe the steps to construct the necessary strains and to optimize auxin-inducible depletion in Saccharomyces cerevisiae. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Construction of TIR1-expressing strains by transformation Basic Protocol 2: Tagging a yeast protein of interest with an auxin-inducible degron Support Protocol: Construction of depletion strains by genetic crosses Basic Protocol 3: Optimization for depletion of the auxin-inducible-degron-tagged protein.
Assuntos
Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas F-Box/metabolismo , Proteólise , Receptores de Superfície Celular/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Mechanisms for coregulation of transcription of tandem genes in yeast remain largely speculative. This study focused on inositol-mediated regulation of the tandem gene pair SNA3-INO1. While the pattern of regulation of these two genes was similar, results showed that intermediate levels of inositol repressed INO1 and induced SNA3. Results also showed that inositol-mediated regulation of the SNA3 gene was not a function of its promoter but occurred from factors within the SNA3-INO1 intergenic region. The basic helix-loop-helix proteins, Ino2p and Ino4p, mediated this regulation through the upstream activation sequence (UAS)(INO) (E-box) sequences in the intergenic region. These results provide a model for studying coregulation of yeast tandem genes. This is especially significant given that many tandem gene pairs in yeast are coregulated even though context-specific UAS sequences are known only for one gene in the pair.