Accurate initiation of mRNA synthesis in extracts from Schizosaccharomyces pombe, Kluyveromyces lactis and Candida glabrata.
We demonstrate the successful adaptation to other yeast species of a protocol previously described for production of transcriptionally active whole cell extracts from Saccharomyces cerevisiae (Woontner and Jaehning, 1990, J. Biol. Chem. 265, 8979-8982). Extracts prepared from Schizosaccharomyces pombe, Kluyveromyces lactis and Candida glabrata were all capable of initiating transcription from a template containing the S. cerevisiae CYC1 TATA box fused to a G-less cassette. Transcription in all of the extracts was sensitive to inhibition by alpha-amanitin, indicating that it was catalysed by RNA polymerase II, and was dramatically stimulated by the chimeric activator GAL4/VP16. The different extracts used different subsets of a group of three initiation sites.
Woontner
M
M
Department of Biology, Indiana University, Bloomington 47405.
Jaehning
J A
JA
D000546:Amanitins / Q000494:pharmacology; D002175:Candida / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D005800:Genes, Fungal; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D006163:Ribonuclease T1 / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D016385:TATA Box; D015534:Trans-Activators / Q000494:pharmacology; D014157:Transcription Factors; D014158:Transcription, Genetic; D015003:Yeasts / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D000546:Amanitins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C070633:Gal-VP16; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D006163:Ribonuclease T1