RESUMO
In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Ribossômicas/genética , Schizosaccharomyces/genética , TATA Box , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Expressão Gênica , Complexo Mediador/genética , Complexo Mediador/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Schizosaccharomyces/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIIA/genética , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismo , Transcrição GênicaRESUMO
The region in promoters that specifies the transcription machinery is called the core promoter, displaying core promoter elements (CPE) necessary for establishment of a preinitiation complex and the initiation of transcription. A classical CPE is the TATA box. In fission yeast, Schizosaccharomyces pombe, a new CPE, called HomolD box, was discovered. Collectively, 141 ribosomal protein genes encoding the full set of 79 different ribosomal proteins and more than 60 other housekeeping genes display a HomolD box in the core promoter. Here, we show that transcription directed by the HomolD box requires the RNA polymerase II machinery, including the general transcription factors. Most intriguingly, however, we identify, by DNA affinity purification, Rrn7 as the protein binding to the HomolD box. Rrn7 is an evolutionary conserved member of the RNA polymerase I machinery involved in transcription initiation of core ribosomal DNA promoters. ChIP shows that Rrn7 cross-links to a ribosomal protein gene promoter containing the HomolD box but not to a promoter containing a TATA box. Taken together, our results suggest that Rrn7 is an excellent candidate to be involved in the coordination of ribosomal DNA and ribosomal gene transcription during ribosome synthesis and, therefore, offer a new perspective to study conservation and evolvability of regulatory networks in eukaryotes.