Isolation of genomic DNA encoding transcription factor TFIID from Acanthamoeba castellanii: characterization of the promoter

• Published in: Nucleic acids research Vol. 20; no. 18; pp. 4817 - 4824
• Main Authors: Wong, Jie Min, Liu, Feng, Baterman, Erik
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 25.09.1992
• Subjects: Acanthamoeba - genetics; Acanthamoeba castellanii; Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Cell Nucleus - metabolism; DNA - genetics; DNA - isolation & purification; Exons; Fundamental and applied biological sciences. Psychology; Genes. Genome; Genomic Library; Introns; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis; Promoter Regions, Genetic; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Deletion; TATA Box; Transcription Factor TFIID; Transcription Factors - genetics; Transcription, Genetic; In vitro transcription; Protozoa; Gene; Nucleotide sequence; Transcription promoter; Rhizoflagellata; Deletion; Gene organization; Acanthamoeba castellanii; Structure function relationship
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/20.18.4817

We have isolated a genomic clone encoding Acanthamoeba castellanii TFIID. The clone contains the entire TFIID gene, 300 bp of 5′ promoter sequences and several hundred base pairs of 3′ non-coding sequence. The coding region is Interrupted by two short introns, but is otherwise identical to Acanthamoeba TFIID cDNA. Comparisons between forty four Acanthamoeba intron 5′ and 3′ boundaries suggest a 5′ splice site consensus of GTACG(T/C) and a 3′ consensus of CAG. We determined the position of the transcription initiation site used in vivo, and show that the same site is used in vitro by homologous nuclear extracts. Deletion analysis of the promoter region shows that the minimal promoter required for efficient expression in vitro is located between −97 and +4 relative to the transcription start site. Three regions within the promoter are important for transcription in vitro; sequences between -97 and -35, the TATAAA box and the initiation region. The initiation region is dispensable but appears to position the transcription start site relative to the TATAAA box. The TATAAA box is absolutely required for transcription initiation whereas the upstream region stimulates transcription approximately five-fold.