Dataset Information

Yeast TATA-box transcription factor gene.

ABSTRACT: The first step in the transcription of most protein-encoding genes in eukaryotes is the binding of a transcription factor to the TATA-box promoter element. This TATA-box transcription factor was purified from extracts of the yeast Saccharomyces cerevisiae by using reconstitution of in vitro transcription reactions as an assay. The activity copurified with a protein whose sodium dodecyl sulfate/polyacrylamide gel mobility is 25 kDa. The sequence of the amino-terminal 21 residues of this protein was determined by sequential Edman degradation. A yeast genomic library was screened with mixed oligonucleotides encoding six residues of the protein sequence. The yeast TATA-box factor gene was cloned, and DNA sequencing revealed a 720-base-pair open reading frame encoding a 27,016-Da protein. The identity of the clone was confirmed by expressing the gene in Escherichia coli and detecting TATA-box factor DNA binding and transcriptional activities in extracts of the recombinant E. coli. The TATA-box factor gene was mapped to chromosome five of S. cerevisiae. RNA blot hybridization and nuclease S1 analysis indicated that the major TATA-box factor mRNA is 1.3 kilobases, including an unusually long 5' untranslated region of 188 +/- 5 nucleotides. Homology searches showed a region of distant similarity to the calcium-binding structures of calpains, a structure that has a conformation similar to the helix-turn-helix motif of DNA binding proteins.

SUBMITTER: Schmidt MC

PROVIDER: S-EPMC298155 | biostudies-other | 1989 Oct

REPOSITORIES: biostudies-other

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Yeast TATA-box transcription factor gene.

Schmidt M C MC Kao C C CC Pei R R Berk A J AJ

Proceedings of the National Academy of Sciences of the United States of America 19891001 20

The first step in the transcription of most protein-encoding genes in eukaryotes is the binding of a transcription factor to the TATA-box promoter element. This TATA-box transcription factor was purified from extracts of the yeast Saccharomyces cerevisiae by using reconstitution of in vitro transcription reactions as an assay. The activity copurified with a protein whose sodium dodecyl sulfate/polyacrylamide gel mobility is 25 kDa. The sequence of the amino-terminal 21 residues of this protein w ...[more]

PMID: 2682626

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Project description:Despite being one of the first eukaryotic transcriptional regulatory elements identified, the sequence of a native TATA box and its significance remain elusive. Applying criteria associated with TATA boxes we queried several Saccharomyces genomes and arrived at the consensus TATA(A/T)A(A/T)(A/G). Approximately 20% of yeast genes contain a TATA box. Strikingly, TATA box-containing genes are associated with responses to stress, are highly regulated, and preferentially utilize SAGA rather than TFIID when compared to TATA-less promoters. Transcriptional regulation in yeast appears to be mechanistically bipolar, possibly reflecting a need to balance inducible stress-related responses with constitutive housekeeping functions. A strain containing amino terminal HA-tagged TBP and its parental untagged counterpart BY4741 (Resgen) were grown at 23?C in CSM to OD600 = 0.8-1.0. Cultures were shifted to 37?C for 5 minutes (to mimic heat treatments used elsewhere (Huisinga and Pugh, 2004)), then crosslinked at 23?C with 1% formaldehyde. ChIP was performed as described previously (Strahl-Bolsinger et al., 1997) with the some modification. Following cell disruption with glass beads, the chromatin was partially purified by centrifugation (Kurdistani and Grunstein, 2003). HA-TBP was immunopurified with 12CA5 antibody. After elution and reversal of the crosslinks, samples were subjected to non-specific amplification, labeling, and array hybridization as described (Bohlander et al., 1992; Chitikila et al., 2002). The amplification procedure was modified as follows. Following 15 round B amplification cycles, DNA was purified via QIAquick PCR Purification kit. Ten cycles were used in round C. Intergenic microarrays were generated by PCR amplification of entire intergenic regions of all yeast genes, then spotted onto glass slides. Spot intensities were filtered to remove any signal that was less then one standard deviation above local background. Three replicates were performed and their log2 ratios (enriched/input) were averaged. Keywords = Chromatin Immunoprecipitation Keywords = genome-wide binding Keywords: other

Dataset Information

Yeast TATA-box transcription factor gene.

Publications

Yeast TATA-box transcription factor gene.

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